G-protein coupled receptor polynucleotides and methods of use thereof

ABSTRACT

The present invention describes human G-protein coupled receptors (GPCRs) and their encoding polynucleotides. Also described are expression vectors, host cells, antisense molecules, and antibodies associated with the GPCR polynucleotides and/or polypeptides of this invention. In addition, methods for treating, diagnosing, preventing, and screening for disorders or diseases associated with abnormal biological activity of GPCR are described, as are methods for screening for modulators, e.g., agonists or antagonists, of GPCR activity and/or function.

[0001] This application claims benefit to provisional application U.S.Serial No. 60/313,658 filed Aug. 20, 2001; to provisional applicationU.S. Serial No. 60/340,703, filed Oct. 30, 2001; to provisionalapplication U.S. Serial No. 60/318,675, filed Sep. 12, 2001; toprovisional application U.S. Serial No. 60/355,596, filed Feb. 6, 2002;to provisional application U.S. Serial No. 60/333,417, filed Nov. 26,2001; and to provisional application U.S. Serial No. 60/338,367, filedDec. 6, 2001. The entire teachings of the referenced applications areincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to novel G-protein coupled receptor(GPCR) nucleic acid or polynucleotide sequences (“genes”) which encodeGPCR proteins. This invention further relates to fragments of novel GPCRnucleic acid sequences and their encoded amino acid sequences.Additionally, the invention relates to methods of using the GPCRpolynucleotide sequences and encoded GPCR proteins for genetic screeningand for the treatment of diseases, disorders, conditions, or syndromesassociated with GPCRs.

BACKGROUND OF THE INVENTION

[0003] Many medically significant biological processes that are mediatedby proteins participating in signal transduction pathways involvingG-proteins and/or second messengers, e.g., cAMP, have been established(Lefkowitz, Nature, 351:353-354 (1991)). These proteins are referred toherein as proteins participating in pathways with G-proteins or PPGproteins. Some examples of these proteins include the G protein-coupledreceptors (GPCR), such as those for adrenergic agents and dopamine(Kobilka, B. K., et al., PNAS, 84:46-50 (1987); Kobilka, B. K., et al.,Science, 238:650-656 (1987); Bunzow, J. R., et al., Nature, 336:783-787(1988)), G-proteins themselves, effector proteins, e.g., phospholipaseC, adenylate cyclase, and phosphodiesterase, and actuator proteins,e.g., protein kinase A and protein kinase C (Simon, M. I., et al.,Science, 252:802-8 (1991)).

[0004] For example, in one form of signal transduction, the effect ofhormone binding results in activation of the enzyme adenylate cyclaseinside the cell. Enzyme activation by hormones is dependent on thepresence of the nucleotide GTP, where GTP also influences hormonebinding. A G-protein binds the hormone receptors to adenylate cyclase.The G-protein has further been shown to exchange GTP for bound GDP whenactivated by hormone receptors. The GTP-carrying form then binds to anactivated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by theG-protein itself, returns the G-protein to its basal, inactive form.Thus, the G-protein serves a dual role—as an intermediate that relaysthe signal from receptor to effector, and as a “clock” that controls theduration of the signal.

[0005] The membrane protein gene superfamily of G-protein coupledreceptors (GPCRs) has been characterized as having seven putativetransmembrane domains. The domains are believed to representtransmembrane α-helices connected by extracellular or cytoplasmic loops.GPCRs include a wide range of biologically active receptors, such ashormone, viral, growth factor, and neuronal receptors.

[0006] GPCRs are further characterized as having seven conservedhydrophobic stretches of about 20 to 30 amino acids, connecting at leasteight divergent hydrophilic loops. The G-protein family of coupledreceptors includes dopamine receptors, which bind to neuroleptic drugs,used for treating psychotic and neurological disorders. Other examplesof members of this family of receptors include calcitonin, adrenergic,endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin,histamine, thrombin, kinin, follicle stimulating hormone, opsins,endothelial differentiation gene-1 receptor, rhodopsins, odorant andcytomegalovirus receptors, etc.

[0007] Most GPCRs have single conserved cysteine residues in each of thefirst two extracellular loops which form disulfide bonds that arebelieved to stabilize functional protein structure. The 7 transmembraneregions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 hasbeen implicated in signal transduction. Phosphorylation and lipidation(palmitylation or farnesylation) of cysteine residues can influencesignal transduction of some GPCRs. Most GPCRs contain potentialphosphorylation sites within the third cytoplasmic loop and/or thecarboxyl terminus.

[0008] For several GPCRs, such as the β-adrenoreceptor, phosphorylationby protein kinase A and/or specific receptor kinases mediates receptordesensitization. For some receptors, the ligand binding sites of GPCRsare believed to comprise a hydrophilic socket formed by thetransmembrane domains of several GPCRs. This socket is surrounded byhydrophobic residues of the GPCRs. The hydrophilic side of each GPCRtransmembrane helix is postulated to face inward and form the polarligand-binding site. TM3 has been implicated in several GPCRs as havinga ligand-binding site, which includes the TM3 aspartate residue.Additionally, TM5 serines, a TM6 asparagine and TM6 or TM7phenylalanines or tyrosines are also implicated in ligand binding.

[0009] GPCRs can be intracellularly coupled by heterotrimeric G-proteinsto various intracellular enzymes, ion channels and transporters (see,Johnson et al., Endoc., Rev., 10:317-331(1989)). Different G-proteinβ-subunits preferentially stimulate particular effectors to modulatevarious biological functions in a cell. Phosphorylation of cytoplasmicresidues of GPCRs have been identified as an important mechanism for theregulation of G-protein coupling of some GPCRs. GPCRs are found innumerous sites within a mammalian host.

[0010] GPCRs are one of the largest receptor superfamilies known. Thesereceptors are biologically important and malfunction of these receptorsresults in diseases such as Alzheimer's, Parkinson, diabetes, dwarfism,color blindness, retinal pigmentosa and asthma. GPCRs are also involvedin depression, schizophrenia, sleeplessness, hypertension, anxiety,stress, renal failure and in several other cardiovascular, metabolic,neural, oncology and immune disorders (F. Horn and G. Vriend, J. Mol.Med., 76: 464-468 (1998)). They have also been shown to play a role inHIV infection (Y. Feng et al., Science, 272: 872-877 (1996)). Thestructure of GPCRs consists of seven transmembrane helices that areconnected by loops. The N-terminus is always extracellular andC-terminus is intracellular. GPCRs are involved in signal transduction.The signal is received at the extracellular N-terminus side. The signalcan be an endogenous ligand, a chemical moiety or light. This signal isthen transduced through the membrane to the cytosolic side where aheterotrimeric protein G-protein is activated which in turn elicits aresponse (F. Horn et al., Recept. and Chann., 5: 305-314 (1998)).Ligands, agonists and antagonists, for these GPCRs are used fortherapeutic purposes.

SUMMARY OF THE INVENTION

[0011] The present invention provides GPCR polynucleotides, preferablyfull-length, and their encoded polypeptides. The GPCR polynucleotidesand polypeptides, may be involved in a variety of diseases, disordersand conditions associated with GPCR activity. More specifically, thepresent invention is concerned with the modulation of these GPCRpolynucleotides and encoded products, particularly in providingtreatments and therapies for relevant diseases. Antagonizing orinhibiting the action of the GPCR polynucleotides and polypeptides isespecially encompassed by the present invention.

[0012] It is an object of this invention to provide isolated GPCRpolynucleotides as depicted in SEQ ID NOs:1-13. Another object of thisinvention is to provide GPCR polypeptides, encoded by the polynucleotideof SEQ ID NOs:1-13 and having the encoded amino acid sequences of SEQ IDNOs:14-26, or a functional or biologically active portion of thesesequences.

[0013] It is yet another object of the invention to provide compositionscomprising the GPCR polynucleotide sequences, or fragments thereof, orthe encoded GPCR polypeptides, or fragments or portions thereof. Inaddition, this invention provides pharmaceutical compositions comprisingat least one GPCR polypeptide, or functional portion thereof, whereinthe compositions further comprise a pharmaceutically and physiologicallyacceptable carrier, excipient, or diluent.

[0014] A further embodiment of this invention presents polynucleotidesequences comprising the complement of SEQ ID NOs:1-13, or variantsthereof. In addition, an object of the invention encompasses variationsor modifications of the GPCR sequences which are a result of degeneracyof the genetic code, where the polynucleotide sequences can hybridizeunder moderate or high stringency conditions to the polynucleotidesequences of SEQ ID NOs:1-13.

[0015] It is another object of the invention to provide nucleic acidsequences encoding the novel GPCR polypeptides and antisense of thenucleic acid sequences, as well as oligonucleotides, fragments, orportions of the nucleic acid molecules or antisense molecules. Alsoprovided are expression vectors and host cells comprisingpolynucleotides that encode the GPCR polypeptides.

[0016] A further object of the present invention encompasses amino acidsequences encoded by the novel GPCR nucleic acid sequences. The aminoacid sequences of SEQ ID NOs:14-26 are encoded by the nucleic acidsequences SEQ ID NOs:1-13, respectively. More specifically, these GPCRpolypeptides are of several types, namely, sensory GPCRs, orphan GPCRs,chemokine GPCRs, or very large GPCRs.

[0017] GPCRs have been described in relation to dopamine receptors,rhodopsin receptors, kinin receptors, N-formyl peptide receptors, opioidreceptors, calcitonin receptors, adrenergic receptors, endothelinreceptors, cAMP receptors, adenosine receptors, muscarinic receptors,acetylcholine receptors, serotonin receptors, histamine receptors,thrombin receptors, follicle stimulating hormone receptors, opsinreceptors, endothelial differentiation gene-I receptors, odorantreceptors, and cytomegalovirus receptors.

[0018] In yet another object, the present invention providespharmaceutical compositions comprising the GPCR polynucleotidesequences, or fragments thereof, or the encoded GPCR polypeptidesequences, or fragments or portions thereof. Also provided arepharmaceutical compositions comprising GPCR polypeptide sequences,homologues, or one or more functional portions thereof, wherein thecompositions further comprise a pharmaceutically- and/orphysiologically-acceptable carrier, excipient, or diluent. All fragmentsor portions of the GPCR polynucleotides and polypeptides are preferablyfunctional or active.

[0019] Another object of the invention is to provide methods forproducing a polypeptide comprising the amino acid sequences of SEQ IDNOs:14-26, or a fragment thereof, preferably, a functional fragment orportion thereof, comprising the steps of a) cultivating a host cellcontaining an expression vector containing at least a functionalfragment of the polynucleotide sequence encoding the GPCR proteinsaccording to this invention under conditions suitable for the expressionof the polypeptide; and b) recovering the polypeptide from the hostcell.

[0020] Another object of this invention is to provide a substantiallypurified modulator, preferably an antagonist or inhibitor, of one ormore of the GPCR polypeptides having SEQ ID NOs:14-26. In this regard,and by way of example, a purified antibody, or antigenic epitope thereofthat binds to a polypeptide comprising the amino acid sequence of SEQ IDNOs:14-26, or homologue encoded by a polynucleotide having a nucleicacid sequence, or degenerate thereof, as set forth in any one of SEQ IDNOs:1-13 is provided.

[0021] It is yet another object of the present invention to provide GPCRnucleic acid sequences, polypeptides, peptides and antibodies for use inthe diagnosis and/or screening of disorders or diseases associated withexpression of one or more of the GPCR polynucleotides and their encodedpolypeptide products as described herein. Another object of thisinvention is to provide diagnostic probes or primers for detectingGPCR-related diseases and/or for monitoring a patient's response totherapy. The probe or primer sequences comprise nucleic acid or aminoacid sequences of the GPCRs described herein.

[0022] It is another object of the present invention to provide a methodfor detecting a polynucleotide that encodes a described GPCR polypeptidein a biological sample comprising the steps of: a) hybridizing thecomplement of the polynucleotide sequence encoding SEQ ID NOs:1-13 tothe nucleic acid material of a biological sample, thereby forming ahybridization complex; and b) detecting the hybridization complex,wherein the presence of the complex correlates with the presence of apolynucleotide encoding a GPCR polypeptide in the biological sample. Thenucleic acid material may be further amplified by the polymerase chainreaction prior to hybridization.

[0023] Another object of this invention is to provide methods forscreening for agents which modulate GPCR polypeptides, e.g., agonistsand antagonists, particularly those that are obtained from the screeningmethods as described. As yet a further object, the invention providesmethods for detecting genetic predisposition, susceptibility andresponse to therapy of various GPCR-related diseases, disorders, orconditions.

[0024] It is another object of the present invention to provide methodsfor the treatment or prevention of several GPCR-associated diseases ordisorders including, but not limited to, cancers, and/or cardiovascular,immune, or neurological diseases or disorders. The methods involveadministering to an individual in need of such treatment or preventionan effective amount of a purified antagonist of one or more of GPCRpolypeptide.

[0025] It is yet another object of this invention to provide diagnostickits for the determination of the nucleotide sequences of human GPCRalleles. The kits can comprise reagents and instructions foramplification-based assays, nucleic acid probe assays, protein nucleicacid probe assays, antibody assays or any combination thereof. Such kitsare suitable for screening and the diagnosis of disorders associatedwith aberrant or uncontrolled cellular development and with theexpression of one or more GPCR polynucleotide and encoded GPCRpolypeptide as described herein. Further objects, features, andadvantages of the present invention will be better understood upon areading of the detailed description of the invention when considered inconnection with the accompanying figures or drawings.

[0026] The invention further relates to a polynucleotide encoding apolypeptide fragment of SEQ ID NO:20, 23, and/or 26, or a polypeptidefragment encoded by the cDNA sequence included in the deposited clone,which is hybridizable to SEQ ID NO:7, 10, and/or 13.

[0027] The invention further relates to a polynucleotide encoding apolypeptide domain of SEQ ID NO:20, 23, and/or 26 or a polypeptidedomain encoded by the cDNA sequence included in the deposited clone,which is hybridizable to SEQ ID NO:7, 10, and/or 13.

[0028] The invention further relates to a polynucleotide encoding apolypeptide epitope of SEQ ID NO:20, 23, and/or 26 or a polypeptideepitope encoded by the cDNA sequence included in the deposited clone,which is hybridizable to SEQ ID NO:7, 10, and/or 13.

[0029] The invention further relates to a polynucleotide encoding apolypeptide of SEQ ID NO:20, 23, and/or 26 or the cDNA sequence includedin the deposited clone, which is hybridizable to SEQ ID NO:7, 10, and/or13, having biological activity.

[0030] The invention further relates to a polynucleotide which is avariant of SEQ ID NO:7, 10, and/or 13.

[0031] The invention further relates to a polynucleotide which is anallelic variant of SEQ ID NO:7, 10, and/or 13.

[0032] The invention further relates to a polynucleotide which encodes aspecies homologue of the SEQ ID NO:20, 23, and/or 26.

[0033] The invention further relates to a polynucleotide whichrepresents the complimentary sequence (antisense) of SEQ ID NO:7, 10,and/or 13.

[0034] The invention further relates to a polynucleotide capable ofhybridizing under stringent conditions to any one of the polynucleotidesspecified herein, wherein said polynucleotide does not hybridize understringent conditions to a nucleic acid molecule having a nucleotidesequence of only A residues or of only T residues.

[0035] The invention further relates to an isolated nucleic acidmolecule of SEQ ID NO:20, 23, and/or 26, wherein the polynucleotidefragment comprises a nucleotide sequence encoding a GPCR protein.

[0036] The invention further relates to an isolated nucleic acidmolecule of SEQ ID NO:7, 10, and/or 13, wherein the polynucleotidefragment comprises a nucleotide sequence encoding the sequenceidentified as SEQ ID NO:20, 23, and/or 26 or the polypeptide encoded bythe cDNA sequence included in the deposited clone, which is hybridizableto SEQ ID NO:7, 10, and/or 13.

[0037] The invention further relates to an isolated nucleic acidmolecule of of SEQ ID NO:7, 10, and/or 13, wherein the polynucleotidefragment comprises the entire nucleotide sequence of SEQ ID NO:7, 10,and/or 13 or the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:7, 10, and/or 13.

[0038] The invention further relates to an isolated nucleic acidmolecule of SEQ ID NO:7, 10, and/or 13, wherein the nucleotide sequencecomprises sequential nucleotide deletions from either the C-terminus orthe N-terminus.

[0039] The invention further relates to an isolated polypeptidecomprising an amino acid sequence that comprises a polypeptide fragmentof SEQ ID NO:20, 23, and/or 26 or the encoded sequence included in thedeposited clone.

[0040] The invention further relates to a polypeptide fragment of SEQ IDNO:20, 23, and/or 26 or the encoded sequence included in the depositedclone, having biological activity.

[0041] The invention further relates to a polypeptide domain of SEQ IDNO:20, 23, and/or 26 or the encoded sequence included in the depositedclone.

[0042] The invention further relates to a polypeptide epitope of SEQ IDNO:20, 23, and/or 26 or the encoded sequence included in the depositedclone.

[0043] The invention further relates to a full length protein of SEQ IDNO:20, 23, and/or 26 or the encoded sequence included in the depositedclone.

[0044] The invention further relates to a variant of SEQ ID NO:20, 23,and/or 26.

[0045] The invention further relates to an allelic variant of SEQ IDNO:20, 23, and/or 26. The invention further relates to a specieshomologue of SEQ ID NO:20, 23, and/or 26.

[0046] The invention further relates to the isolated polypeptide of ofSEQ ID NO:20, 23, and/or 26, wherein the full length protein comprisessequential amino acid deletions from either the C-terminus or theN-terminus.

[0047] The invention further relates to an isolated antibody that bindsspecifically to the isolated polypeptide of SEQ ID NO:20, 23, and/or 26.

[0048] The invention further relates to a method for preventing,treating, or ameliorating a medical condition, comprising administeringto a mammalian subject a therapeutically effective amount of thepolypeptide of SEQ ID NO:20, 23, and/or 26 or the polynucleotide of SEQID NO:7, 10, and/or 13.

[0049] The invention further relates to a method of diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising the steps of (a) determining the presence orabsence of a mutation in the polynucleotide of SEQ ID NO:7, 10, and/or13; and (b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.

[0050] The invention further relates to a method of diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising the steps of (a) determining the presence oramount of expression of the polypeptide of SEQ ID NO:20, 23, and/or 26in a biological sample; and diagnosing a pathological condition or asusceptibility to a pathological condition based on the presence oramount of expression of the polypeptide.

[0051] The invention further relates to a method for identifying abinding partner to the polypeptide of SEQ ID NO:20, 23, and/or 26comprising the steps of (a) contacting the polypeptide of SEQ ID NO:20,23, and/or 26 with a binding partner; and (b) determining whether thebinding partner effects an activity of the polypeptide.

[0052] The invention further relates to a gene corresponding to the cDNAsequence of SEQ ID NO:7, 10, and/or 13. The invention further relates toa method of identifying an activity in a biological assay, wherein themethod comprises the steps of expressing SEQ ID NO:7, 10, and/or 13 in acell, (b) isolating the supernatant; (c) detecting an activity in abiological assay; and (d) identifying the protein in the supernatanthaving the activity.

[0053] The invention further relates to a process for makingpolynucleotide sequences encoding gene products having altered SEQ IDNO:20, 23, and/or 26 activity comprising the steps of (a) shuffling anucleotide sequence of SEQ ID NO:7, 10, and/or 13, (b) expressing theresulting shuffled nucleotide sequences and, (c) selecting for alteredactivity as compared to the activity of the gene product of saidunmodified nucleotide sequence.

[0054] The invention further relates to a shuffled polynucleotidesequence produced by a shuffling process, wherein said shuffled DNAmolecule encodes a gene product having enhanced tolerance to aninhibitor of SEQ ID NO:20, 23, and/or 26 activity.

[0055] The invention further relates to a method of identifying acompound that modulates the biological activity of Gene 7, 10, and/or13, comprising the steps of, (a) combining a candidate modulatorcompound with Gene 7, 10, and/or 13 having the sequence set forth in oneor more of SEQ ID NO:20, 23, and/or 26; and measuring an effect of thecandidate modulator compound on the activity of Gene 7, 10, and/or 13.

[0056] The invention further relates to a method of identifying acompound that modulates the biological activity of a GPCR, comprisingthe steps of, (a) combining a candidate modulator compound with a hostcell expressing Gene 7, 10, and/or 13 having the sequence as set forthin SEQ ID NO:20, 23, and/or 26; and, (b) measuring an effect of thecandidate modulator compound on the activity of the expressed Gene 7,10, and/or 13.

[0057] The invention further relates to a method of identifying acompound that modulates the biological activity of Gene 7, 10, and/or13, comprising the steps of, (a) combining a candidate modulatorcompound with a host cell containing a vector described herein, whereinGene 7, 10, and/or 13 is expressed by the cell; and, (b) measuring aneffect of the candidate modulator compound on the activity of theexpressed Gene 7, 10, and/or 13.

[0058] The invention further relates to a method of screening for acompound that is capable of modulating the biological activity of Gene7, 10, and/or 13, comprising the steps of: (a) providing a host celldescribed herein; (b) determining the biological activity of Gene 7, 10,and/or 13 in the absence of a modulator compound; (c) contacting thecell with the modulator compound; and (d) determining the biologicalactivity of Gene 7, 10, and/or 13 in the presence of the modulatorcompound; wherein a difference between the activity of Gene 7, 10,,and/or 13 in the presence of the modulator compound and in the absenceof the modulator compound indicates a modulating effect of the compound.

[0059] The invention further relates to a compound that modulates thebiological activity of human Gene 7, 10, and/or 13 as identified by themethods described herein.

[0060] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:26, in addition to, its encoding nucleic acid,wherein the medical condition is a neural disorder.

[0061] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:26, in addition to, its encoding nucleic acid,wherein the medical condition is an endocrine disorder.

[0062] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:26, in addition to, its encoding nucleic acid,wherein the medical condition is a sleep disorder.

[0063] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:26, in addition to, its encoding nucleic acid,wherein the medical condition is a member of the group consisting ofdisorders that affect the nucleus accumbens, disorders that affect thebrains ‘reward center’ function, neurotransmitter release disorders,disorders affecting the release of dopamine, disorders affecting therelease of opioid peptides, disorders affecting the release ofserotonin, disorders affecting the release of GABA, pineal glanddisorders, disorders affecting the establishment of circadian rhythms,disorders affecting the maintenance of circadian rhythms, disordersaffecting the control of the sleep/wake cycle.

[0064] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:26, in addition to, its encoding nucleic acid,wherein the medical condition is a member of the group consisting ofmelatonin secretion disorders, pituitary hormone secretion disorders,oxytocin secretion disorders, disorders affecting neuroendocrineresponse to stressful stimuli, disorders affecting oxytocin secretionduring neuroendocrine response to stressful stimuli, disorders affectingnocturnal patterns of hormone secretion, disorders affecting thenocturnal hormone secretion of prolactin, disorders affecting thenocturnal hormone secretion of cortisol, and/or disorders affecting thenocturnal hormone secretion of growth hormone.

[0065] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:26, in addition to, its encoding nucleic acid,wherein the medical condition is a member of the group consisting ofneuro-pathologies, including responses to stress, and propensity todevelop addictive behaviors, as well as a vast number of neuroendocrineabnormalities including sleep disorders.

[0066] The present invention also relates to an isolated polynucleotideconsisting of a portion of the human Gene 7 gene consisting of at least8 bases, specifically excluding the polynucleotide sequence provided inGenbank Accession Nos. BG198766; BI828553; BG210740; BE439409; AF003828;and/or AL705589.

[0067] The present invention also relates to an isolated polynucleotideconsisting of a nucleotide sequence encoding a fragment of the humanGene 7 protein, wherein said fragment displays one or more functionalactivities specifically excluding the polynucleotide sequence providedin Genbank Accession Nos. BGI98766; BI828553; BG210740; BE439409;AF003828; and/or AL705589.

[0068] The present invention also relates to the polynucleotide of SEQID NO:7 consisting of at least 10 to 50 bases, wherein said at least 10to 50 bases specifically exclude the polynucleotide sequence of GenbankAccession Nos. BG198766; BI828553; BG210740; BE439409; AF003828; and/orAL705589.

[0069] The present invention also relates to the polynucleotide of SEQID NO:7 consisting of at least 15 to 100 bases, wherein said at least 15to 100 bases specifically exclude the polynucleotide sequence of GenbankAccession Nos. BG198766; BI828553; BG210740; BE439409; AF003828; and/orAL705589.

[0070] The present invention also relates to the polynucleotide of SEQID NO:7 consisting of at least 100 to 1000 bases, wherein said at least100 to 1000 bases specifically exclude the polynucleotide sequence ofGenbank Accession Nos. BG198766; BI828553; BG210740; BE439409; AF003828;and/or AL705589.

[0071] The present invention also relates to an isolated polypeptidefragment of the human Gene 7 protein, wherein said polypeptide fragmentdoes not consist of the polypeptide encoded by the polynucleotidesequence of Genbank Accession Nos. BG198766; BI828553; BG210740;BE439409; AF003828; and/or AL705589.

[0072] The present invention also relates to an isolated polynucleotideconsisting of a portion of the human Gene 10 gene consisting of at least8 bases, specifically excluding the polynucleotide sequence provided inGenbank Accession Nos. BB201968; BB206141); AI962273; and/or BI274717.

[0073] The present invention also relates to an isolated polynucleotideconsisting of a nucleotide sequence encoding a fragment of the humanGene 10 protein, wherein said fragment displays one or more functionalactivities specifically excluding the polynucleotide sequence providedin Genbank Accession Nos. BB201968; BB206141); AI962273; and/orBI274717.

[0074] The present invention also relates to the polynucleotide of SEQID NO:10 consisting of at least 10 to 50 bases, wherein said at least 10to 50 bases specifically exclude the polynucleotide sequence of GenbankAccession Nos. BB201968; BB206141); AI962273; and/or BI274717.

[0075] The present invention also relates to the polynucleotide of SEQID NO:10 consisting of at least 15 to 100 bases, wherein said at least15 to 100 bases specifically exclude the polynucleotide sequence ofGenbank Accession Nos. BB201968; BB206141); AI962273; and/or BI274717.

[0076] The present invention also relates to the polynucleotide of SEQID NO:10 consisting of at least 100 to 1000 bases, wherein said at least100 to 1000 bases specifically exclude the polynucleotide sequence ofGenbank Accession Nos. BB201968; BB206141); AI962273; and/or BI274717.

[0077] The present invention also relates to an isolated polypeptidefragment of the human Gene 10 protein, wherein said polypeptide fragmentdoes not consist of the polypeptide encoded by the polynucleotidesequence of Genbank Accession Nos. BB201968; BB206141); AI962273; and/orBI274717.

[0078] The present invention also relates to an isolated polynucleotideconsisting of a nucleotide sequence encoding a fragment of the humanGene 13 protein, wherein said fragment displays one or more functionalactivities specifically excluding the polynucleotide sequence providedin Genbank Accession Nos. BQ339434; and/or BG003773.

[0079] The present invention also relates to the polynucleotide of SEQID NO:13 consisting of at least 10 to 50 bases, wherein said at least 10to 50 bases specifically exclude the polynucleotide sequence of GenbankAccession Nos. BQ339434; and/or BG003773.

[0080] The present invention also relates to the polynucleotide of SEQID NO:13 consisting of at least 15 to 100 bases, wherein said at least15 to 100 bases specifically exclude the polynucleotide sequence ofGenbank Accession Nos. BQ339434; and/or BG003773.

[0081] The present invention also relates to the polynucleotide of SEQID NO:13 consisting of at least 100 to 1000 bases, wherein said at least100 to 1000 bases specifically exclude the polynucleotide sequence ofGenbank Accession Nos. BQ339434; and/or BG003773.

[0082] The present invention also relates to an isolated polypeptidefragment of the human Gene 13 protein, wherein said polypeptide fragmentdoes not consist of the polypeptide encoded by the polynucleotidesequence of Genbank Accession Nos. BQ339434; and/or BG003773.

BRIEF DESCRIPTION OF THE FIGURES

[0083]FIG. 1 presents the nucleic acid sequence (SEQ ID NO:1) of a novelhuman sensory GPCR, called Gene 1 herein.

[0084]FIG. 2 presents the amino acid sequence (SEQ ID NO:14) encoded bythe nucleic acid sequence (SEQ ID NO:1) of Gene 1.

[0085]FIG. 3 presents the nucleic acid sequence (SEQ ID NO:2) of a novelhuman sensory GPCR, called Gene 2 herein.

[0086]FIG. 4 presents the amino acid sequence (SEQ ID NO:15) encoded bythe nucleic acid sequence (SEQ ID NO:2) of Gene 2.

[0087]FIG. 5 presents the nucleic acid sequence (SEQ ID NO:3) of a novelhuman sensory GPCR, called Gene 3 herein.

[0088]FIG. 6 presents the amino acid sequence (SEQ ID NO:16) encoded bythe nucleic acid sequence (SEQ ID NO:3) of Gene 3.

[0089]FIG. 7 shows the nucleic acid sequence (SEQ ID NO:4) of a novelhuman sensory GPCR, called Gene 4 herein.

[0090]FIG. 8 shows the amino acid sequence (SEQ ID NO:17) encoded by thenucleic acid sequence (SEQ ID NO:4) of Gene 4.

[0091]FIG. 9 shows the nucleic acid sequence (SEQ ID NO:5) of a novelhuman sensory GPCR, called Gene 5 herein.

[0092]FIG. 10 shows the amino acid sequence (SEQ ID NO:18) encoded bythe nucleic acid sequence (SEQ ID NO:5) of Gene 5.

[0093]FIG. 11 shows the nucleic acid sequence (SEQ ID NO:6) of a novelhuman chemokine GPCR, called Gene 6 herein.

[0094]FIG. 12 shows the amino acid sequence (SEQ ID NO:19) encoded bythe nucleic acid sequence (SEQ ID NO:6) of Gene 6.

[0095]FIGS. 13A, 13B, and 13C present the nucleic acid sequence (SEQ IDNO:7) of a novel human orphan GPCR, called Gene 7 herein.

[0096]FIG. 14 presents the amino acid sequence (SEQ ID NO:20) encoded bythe nucleic acid sequence (SEQ ID NO:7) of Gene 7.

[0097]FIGS. 15A and 15B present the nucleic acid sequence (SEQ ID NO:8)of a novel human orphan GPCR, called Gene 8 herein.

[0098]FIG. 16 presents the amino acid sequence (SEQ ID NO:21) encoded bythe nucleic acid sequence (SEQ ID NO:8) of Gene 8.

[0099]FIG. 17 presents the nucleic acid sequence (SEQ ID NO:9) of anovel human sensory GPCR, called Gene 9 herein.

[0100]FIG. 18 presents the amino acid sequence (SEQ ID NO:22) encoded bythe nucleic acid sequence (SEQ ID NO:9) of Gene 9.

[0101] FIGS. 19A-B shows the nucleic acid sequence (SEQ ID NO:10) of anovel human sensory GPCR, called Gene 10 herein.

[0102]FIG. 20 shows the amino acid sequence (SEQ ID NO:23) encoded bythe nucleic acid sequence (SEQ ID NO:10) of Gene 10.

[0103]FIGS. 21A and 21B show the nucleic acid sequence (SEQ ID NO:11) ofa novel human sensory GPCR, called Gene 11 herein.

[0104]FIG. 22 shows the amino acid sequence (SEQ ID NO:24) encoded bythe nucleic acid sequence (SEQ ID NO:11) of Gene 11.

[0105]FIG. 23 presents the nucleic acid sequence (SEQ ID NO:12) of anovel human sensory GPCR, called Gene 12 herein.

[0106]FIG. 24 presents the amino acid sequence (SEQ ID NO:25) encoded bythe nucleic acid sequence (SEQ ID NO:12) of Gene 12.

[0107] FIGS. 25A-B presents the nucleic acid sequence (SEQ ID NO:13) ofa novel human very large GPCR, called Gene 13 herein.

[0108]FIG. 26 presents the amino acid sequence (SEQ ID NO:26) encoded bythe nucleic acid sequence (SEQ ID NO:13) of Gene 13.

[0109]FIG. 27A illustrates an alignment of the novel human sensory GPCRGene 1 with the top hit protein human olfactory receptor 5U1 (GenbankAccession No: gi|14423824; SEQ ID NO:72) a transmembrane receptor) usingthe protein sequence database and BLAST analysis as known and asdescribed herein. FIG. 27B illustrates the domain prediction for theGPCR encoded by Gene 1. (“T” denotes “target”, and represents a portionof the amino acid sequence of Gene 1 provided as SEQ ID NO:14). Domainpredictions are valuable for suggesting possible functional domains inthe predicted protein. These predictions are based on comparisons of thegiven protein sequence (the “query”, or “Q” represents the Pfam PF00007Rhodopsin model sequence provided as SEQ ID NO:86) against a collectionof statistical models known as Hidden Markov Models (HMMs) (the targets,or T). HMMs represent consensus patterns for known functional domainsand this method of comparison allows for the prediction of functionaldomains in novel protein sequences. HMMs are built from the Pfamalignments. The Pfam is a database of multiple alignments of proteindomains or conserved protein regions. The alignments represent someevolutionary conserved structure, which has implications for theprotein's function. Such alignment analysis can be very useful forautomatically recognizing that a new protein belongs to an existingprotein family, even if the homology is weak (See, A. Bateman, E.Birney, R. Durbin, S. R. Eddy, K. L. Howe, and E. L. L. Sonnhammer. ThePfam Protein Families Database. Nucleic Acids Research, 28:263-266,2000).

[0110] In FIG. 27A, the query (or “Q”) sequence is that of Gene 1 (SEQID NO: 14), while the subject (“sbjct”) sequence is that of the sequencehaving the highest percent identity (50%), i.e., human olfactoryreceptor 5U1, for this GPCR sequence (Genbank Accession No: gi|14423824;SEQ ID NO:72).

[0111]FIGS. 28A and 28B illustrate an alignment of the novel humansensory GPCR Gene 2 with the top hit proteins from the protein sequencedatabase and BLAST analysis as known and also as described herein. FIG.28A shows that the GPCR Gene 2 amino acid sequence is highly similar tohuman G protein coupled receptor 61 protein (Genbank Accession No:gi|13994320; SEQ ID NO:73). FIG. 27B shows that the Gene 2 amino acidsequence is also highly similar to rabbit G protein coupled receptorprotein (Genbank Accession No: gi|AAR91232; SEQ ID NO:74). In FIGS. 28A,28B, and 28C, the query (or “Q”) sequence is that of Gene 2 (SEQ IDNO:15); in FIG. 28A, the subject (“sbjct”) sequence(s) is/are the aminoacid sequence(s) having the highest percent identity (98%) to that ofGene 2. FIG. 28C illustrates the predicted domains in the GPCR encodedby Gene 2. (“T” denotes “target” and represents the Pfam PF00007Rhodopsin model sequence provided as SEQ ID NO:86).

[0112]FIG. 29A illustrates an alignment of the novel human sensory GPCRGene 3 with the top hit protein, i.e., MOR 3′Beta4 protein of mouse(Genbank Accession No: gi|11908220; SEQ ID NO:75), from the proteinsequence database and BLAST analysis as known and as described herein.FIG. 29B illustrates the domain prediction in the GPCR encoded by Gene3. (“T” denotes “target” and represents the Pfam PF00007 Rhodopsin modelsequence provided as SEQ ID NO:86). In FIGS. 29A and 29B, the query (or“Q”) sequence is that of Gene 3, while the subject (“sbjct”) sequence isthe amino acid sequence having the highest percent identity (48%) tothat of Gene 3 (Genbank Accession No: gi|11908220; SEQ ID NO:75).

[0113]FIG. 30A illustrates an alignment of the novel human sensory GPCRGene 4 with the top hit protein, i.e., MOR 3′Beta1 protein (olfactoryreceptor 67) of mouse (Genbank Accession No: gi|4761597; SEQ ID NO:76),from the protein sequence database and BLAST analysis as known and asdescribed herein. FIG. 30B illustrates the domain prediction in the GPCRencoded by Gene 4. (“T” denotes “target” and represents the Pfam PF00007Rhodopsin model sequence provided as SEQ ID NO:86). In FIGS. 30A and30B, the query (or “Q”) sequence is that of Gene 4, while the subject(“sbjct”) sequence is the amino acid sequence having the highest percentidentity (48%) to that of Gene 4 (Genbank Accession No: gi|4761597; SEQID NO:76).

[0114]FIG. 31 illustrates an alignment of the novel human sensory GPCRGene 5 with the top hit protein, i.e., human taste receptor protein(Genbank Accession No: gi|7262621; SEQ ID NO:77) from the proteinsequence database and BLAST analysis as known and as described herein.In FIG. 31, the query (or “Q”) sequence is that of Gene 5, while thesubject (“sbjct”) sequence is the amino acid sequence having the highestpercent identity (44%) to that of Gene 5 (Genbank Accession No:gi|7262621; SEQ ID NO:77).

[0115]FIG. 32 illustrates an alignment of the novel human chemokine GPCRGene 6 with the top hit protein, i.e., human chemokine receptor 1(Genbank Accession No: gi|12729981; SEQ ID NO:78) from the proteinsequence database and BLAST analysis as known and as described herein.In FIG. 32, the query (or “Q”) sequence is that of Gene 6, while thesubject (“sbjct”) sequence is the amino acid sequence having the highestpercent identity (26%) to that of Gene 6 (Genbank Accession No:gi|12729981; SEQ ID NO:78).

[0116]FIG. 33 illustrates an alignment of the novel human orphan GPCRGene 7 with the top hit protein, i.e., human G-protein coupled receptorhHI7T213 (Genbank Accession No: gi|AAY90761; SEQ ID NO:79) from theprotein sequence database and BLAST analysis as known and as describedherein. In FIG. 33, the query (or “Q”) sequence is that of Gene 7, whilethe subject (“sbjct”) sequence is the amino acid sequence having thehighest percent identity (77%) to that of Gene 7 (Genbank Accession No:gi|AAY90761; SEQ ID NO:79).

[0117]FIG. 34 illustrates an alignment of the novel human orphan GPCRGene 8 with the top hit protein, i.e., human G-protein coupled receptorRE2 (Genbank Accession No: gi|13637713; SEQ ID NO:80) from the proteinsequence database and BLAST analysis as known and as described herein.In FIG. 34, the query (or “Q”) sequence is that of Gene 8, while thesubject (“sbjct”) sequence is the amino acid sequence having the highestpercent identity (32%) to that of Gene 8 (Genbank Accession No:gi|13637713; SEQ ID NO:80).

[0118]FIG. 35 illustrates an alignment of the novel human sensory GPCRGene 9 with the top hit protein, i.e., human sensory GPCR receptor(Genbank Accession No: gi|3746448; SEQ ID NO:81) from the proteinsequence database and BLAST analysis as known and as described herein.In FIG. 35, the query (or “Q”) sequence is that of Gene 9, while thesubject (“sbjct”) sequence is the amino acid sequence having the highestpercent identity (47%) to that of Gene 9 (Genbank Accession No:gi|3746448; SEQ ID NO:81).

[0119]FIG. 36 illustrates an alignment of the novel human sensory GPCRGene 10 with the top hit protein, i.e., odorant receptor K11 of mouse(Genbank Accession No: gi|11692519; SEQ ID NO:82), from the proteinsequence database and BLAST analysis as known and as described herein.In FIG. 36, the query (or “Q”) sequence is that of Gene 10, while thesubject (“sbjct”) sequence is the amino acid sequence having the highestpercent identity (76%) to that of Gene 10 (Genbank Accession No:gi|11692519; SEQ ID NO:82).

[0120]FIG. 37 illustrates an alignment of the novel human sensory GPCRGene 11 with the top hit protein, i.e., odorant receptor K4h11 of mouse(Genbank Accession No: gi|11692563; SEQ ID NO:83), from the proteinsequence database and BLAST analysis as known and as described herein.In FIG. 37, the query (or “Q”) sequence is that of Gene 11, while thesubject (“sbjct”) sequence is the amino acid sequence having the highestpercent identity (78%) to that of Gene 11 (Genbank Accession No:gi|11692563; SEQ ID NO:83).

[0121]FIG. 38 illustrates an alignment of the novel human sensory GPCRGene 12 with the top hit protein, i.e., vomeronasal receptor V1RC3 ofmouse (Genbank Accession No: gi|11967419; SEQ ID NO:84), from theprotein sequence database and BLAST analysis as known and as describedherein. In FIG. 38, the query (or “Q”) sequence is that of Gene 12,while the subject (“sbjct”) sequence is the amino acid sequence havingthe highest percent identity (43%) to that of Gene 12 (Genbank AccessionNo: gi|11967419; SEQ ID NO:84).

[0122]FIG. 39 illustrates an alignment of the novel the human very largeGPCR Gene 13 with the top hit protein, i.e., human very large G-proteincoupled receptor-1 (Genbank Accession No: gi|5902966; SEQ ID NO:85),from the protein sequence database and BLAST analysis as known and asdescribed herein. In FIG. 39, the query (or “Q”) sequence is that ofGene 13, while the subject (“sbjct”) sequence is the amino acid sequencehaving the highest percent identity (30%) to that of Gene 13 (GenbankAccession No: gi|5902966; SEQ ID NO:85).

[0123] FIGS. 40A-40E illustrate a multiple sequence alignment of theamino acid sequence of GPCR, Gene 13, (SEQ ID NO:26) with the amino acidsequences of other human GPCR proteins, namely, human_hypothetical 1(SEQ ID NO:149) and human_hypothetical 2 (SEQ ID NO:150). The GCG pileupprogram was used to generate the alignment. The blackened areasrepresent identical amino acids in more than half of the listedsequences and the gray highlighted areas represent similar amino acids.Dashes represent no comparison and dots represent gaps in the alignment.

[0124]FIG. 41 presents the tissue expression profile of the novel humanGPCR, Gene 13. A PCR primer was designed from SEQ ID NO:13 and was usedto measure the steady state levels of mRNA by quantitative PCR.Transcripts corresponding to the GPCR, Gene 13, is highly expressed inbrain tissues and the pituitary.

[0125]FIG. 42 presents the brain sub-region expression profile of thenovel human GPCR, Gene 13. A PCR primer was designed from SEQ ID NO:13and was used to measure the steady state levels of mRNA by quantitativePCR. Transcripts corresponding to the GPCR, HGPRBMY 37, is highlyexpressed in the following brain sub-regions: amygdala, cerebellum,corpus callosum, caudate nucleus, hippocampus, subtantia nigra andthalamus.

[0126]FIG. 43 presents a schematic of the cell-based reporter assaysystem based on Fluorescence Resonance Energy Transfer (FRET) to detectGene 13 functional coupling as described in Example 8. Gene 13 istransfected into the Cho/NFAT-CRE reporter cell line and changes inreal-time gene expression, as a consequence of constitutive G-proteincoupling of Gene 13 GPCR, is examined by analyzing the fluorescenceemission of the transformed cells at 447 nm and 518 nm

[0127]FIG. 44 shows an expanded expression profile of the novelG-protein coupled receptor, Gene 13. The figure illustrates the relativeexpression level of Gene 13 amongst various mRNA tissue sources. Asshown, the Gene 13 polypeptide was expressed predominately in thenervous system, with lesser amounts found in the respiratory andendocrine systems. Specifically, Gene 13 was expressed at the higheststeady state levels in the nucleus accumbens, followed by the pineal andpituitary gland. Expression of Gene 13 was also significantly expressedat near equal levels across the cortex, hippocampus, amygdala, andchoroid plexus. Expression of Gene 13 was also significantly expressedto a lesser extent in in the caudate, the cerebellum and thehypothalamus. Expression data was obtained by measuring the steady stateGene 13 mRNA levels by quantitative PCR using the PCR primer pairprovided as SEQ ID NO:166 and 167, and Taqman probe (SEQ ID NO:168) asdescribed in Example 10 herein.

DETAILED DESCRIPTION OF THE INVENTION

[0128] The present invention provides novel human GPCR (GPCR) genes(i.e., polynucleotide or nucleic acid sequences) which encode GPCRproteins (polypeptides), preferably full-length GPCR polypeptides. Theinvention further relates to fragments and portions of novel GPCRnucleic acid sequences and their encoded amino acid sequences(peptides). Preferably, the fragments and portions of the GPCRpolypeptides are functional or active. The invention also providesmethods of using the novel GPCR polynucleotide sequences and the encodedGPCR polypeptides for genetic screening and for the treatment ofdiseases, disorders, conditions, or syndromes associated with GPCRs andGPCR activity and function.

[0129] Definitions

[0130] The following definitions are provided to more fully describe thepresent invention in its various aspects. The definitions are intendedto be useful for guidance and elucidation, and are not intended to limitthe disclosed invention or its embodiments.

[0131] “Amino acid sequence” as used herein can refer to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, as well as to naturally occurring or syntheticmolecules, preferably isolated polypeptides of the GPCR. Amino acidsequence fragments are typically from about 4 to about 30, preferablyfrom about 5 to about 15, more preferably from about 5 to about 15 aminoacids in length and preferably retain the biological activity orfunction of a GPCR polypeptide. GPCR amino acid sequences of thisinvention are set forth in SEQ ID NOs:14-26 of Table 1 and indescription of the Figures. The terms GPCR polypeptide and GPCR proteinare used interchangeably herein to refer to the encoded products of theGPCR nucleic acid sequences according to the present invention.

[0132] As will be appreciated by the skilled practitioner, should theamino acid fragment comprise an antigenic epitope, for example,biological function per se need not be maintained. The terms Gene 7, 10,and/or 13 polypeptide and Gene 7, 10, and/or 13 protein are usedinterchangeably herein to refer to the encoded product of the Gene 7,10, and/or 13 nucleic acid sequence according to the present invention.

[0133] Isolated GPCR polypeptide refers to the amino acid sequence ofsubstantially purified GPCR, which may be obtained from any species,preferably mammalian, and more preferably, human, and from a variety ofsources, including natural, synthetic, semi-synthetic, or recombinant.More particularly, the GPCR polypeptides of this invention areidentified in SEQ ID NOs:14-26. Functional fragments of the GPCRpolypeptides are also embraced by the present invention.

[0134] “Similar” amino acids are those which have the same or similarphysical properties and in many cases, the function is conserved withsimilar residues. For example, amino acids lysine and arginine aresimilar; while residues such as proline and cysteine do not share anyphysical property and are not considered to be similar. The term“consensus” refers to a sequence that reflects the most common choice ofbase or amino acid at each position among a series of related DNA, RNAor protein sequences. Areas of particularly good agreement oftenrepresent conserved functional domains.

[0135] A “variant” of a GPCR polypeptide refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, in which a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “non-conservative”changes, for example, replacement of a glycine with a tryptophan. Theencoded protein may also contain deletions, insertions, or substitutionsof amino acid residues, which produce a silent change and result in afunctionally equivalent GPCR protein. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues, as long as the biological activityof GPCR protein is retained.

[0136] For example, negatively charged amino acids may include asparticacid and glutamic acid; positively charged amino acids may includelysine and arginine; and amino acids with uncharged polar head groupshaving similar hydrophilicity values may include leucine, isoleucine,and valine; glycine and alanine; asparagine and glutamine; serine andthreonine; and phenylalanine and tyrosine. Guidance in determining whichamino acid residues may be substituted, inserted, or deleted withoutabolishing functional biological or immunological activity may be foundusing computer programs well known in the art, for example, DNASTAR,Inc. software (Madison, Wis.).

[0137] The term “mimetic”, as used herein, refers to a molecule, havinga structure which is developed from knowledge of the structure of a GPCRprotein, or portions thereof, and as such, is able to affect some or allof the actions of the GPCR protein. A mimetic may comprise of asynthetic peptide or an organic molecule.

[0138] “Nucleic acid or polynucleotide sequence”, as used herein, refersto an isolated oligonucleotide (“oligo”), nucleotide, or polynucleotide,and fragments thereof, and to DNA or RNA of genomic or synthetic originwhich may be single- or double-stranded, and represent the sense oranti-sense strand, preferably of the GPCR. By way of non-limitingexamples, fragments include nucleic acid sequences that are greater than20-60 nucleotides in length, and preferably include fragments that areat least 70-100 nucleotides, or which are at least 1000 nucleotides orgreater in length. GPCR nucleic acid sequences of this invention arespecifically identified in SEQ ID NOs:1-13 of Table 1 and as illustratedin the Figures.

[0139] An “allele” or “allelic sequence” is an alternative form of aGPCR nucleic acid sequence. Alleles may result from at least onemutation in a GPCR nucleic acid sequence and may yield altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven gene, whether natural or recombinant, may have none, one, or manyallelic forms. Common mutational changes, which give rise to alleles,are generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0140] “Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide (“oligo”) linked viaan amide bond, similar to the peptide backbone of amino acid residues.PNAs typically comprise oligos of at least 5 nucleotides linked viaamide bonds. PNAs may or may not terminate in positively charged aminoacid residues to enhance binding affinities to DNA. Such amino acidsinclude, for example, lysine and arginine, among others. These smallmolecules stop transcript elongation by binding to their complementarystrand of nucleic acid (P. E. Nielsen et al., 1993, Anticancer DrugDes., 8:53-63). PNA may be pegylated to extend their lifespan in thecell where they preferentially bind to complementary single stranded DNAand RNA.

[0141] “Oligonucleotides” or “oligomers”, as defined herein, refer to aGPCR nucleic acid sequence comprising contiguous nucleotides, of atleast about 5 nucleotides to about 60 nucleotides, preferably at leastabout 8 to 10 nucleotides in length, more preferably at least about 12nucleotides in length, for example, about 15 to 35 nucleotides, or about15 to 25 nucleotides, or about 20 to 35 nucleotides, which can betypically used in PCR amplification assays, hybridization assays, or inmicroarrays. It will be understood that the term oligonucleotide issubstantially equivalent to the terms primer, probe, or amplimer, ascommonly defined in the art. GPCR primers of this invention, (i.e.,left, right, and internal primers), are set forth SEQ ID NOs:27-71 inTables 2 and 3 herein.

[0142] The term “antisense” refers to nucleotide sequences, andcompositions containing nucleic acid sequences, which are complementaryto a specific DNA or RNA sequence. The term “antisense strand” is usedin reference to a nucleic acid strand that is complementary to the“sense” strand. Antisense (i.e., complementary) nucleic acid moleculesinclude PNAs and may be produced by any method, including synthesis ortranscription. Once introduced into a cell, the complementarynucleotides combine with natural sequences produced by the cell to formduplexes, which block either transcription or translation. Thedesignation “negative” is sometimes used in reference to the antisensestrand, and “positive” is sometimes used in reference to the sensestrand.

[0143] “Altered” nucleic acid sequences encoding a GPCR polypeptideinclude nucleic acid sequences containing deletions, insertions and/orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent GPCR polypeptide.Altered nucleic acid sequences may further include polymorphisms of thepolynucleotide encoding a GPCR polypeptide; such polymorphisms may ormay not be readily detectable using a particular oligonucleotide probe.

[0144] The terms “Expressed Sequence Tag” or “EST” refers to the partialsequence of a cDNA insert which has been made by reverse transcriptionof mRNA extracted from a tissue, followed by insertion into a vector asknown in the art (Adams, M. D., et al. Science (1991) 252:1651-1656;Adams, M. D. et al., Nature, (1992) 355:632-634; Adams, M. D., et al.,Nature (1995) 377 Supp:3-174).

[0145] The term “biologically active”, i.e., functional, refers to aprotein or polypeptide or fragment thereof, having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” refers to the capability of anatural, recombinant, or synthetic GPCR, or an oligopeptide thereof, toinduce a specific immune response in appropriate animals or cells, forexample, to generate antibodies, to bind with specific antibodies,and/or to elicit a cellular immune response.

[0146] An “agonist” refers to a molecule which, when bound to, orassociated with, a GPCR polypeptide, or a functional fragment thereof,increases or prolongs the duration of the effect of the GPCRpolypeptide. Agonists may include proteins, nucleic acids,carbohydrates, or any other molecules that bind to and modulate theeffect of GPCR polypeptide. Agonists typically enhance, increase, oraugment the function or activity of a GPCR molecule.

[0147] An “antagonist” refers to a molecule which, when bound to, orassociated with, a GPCR polypeptide, or a functional fragment thereof,decreases the amount or duration of the biological or immunologicalactivity of GPCR polypeptide. Antagonists may include proteins, nucleicacids, carbohydrates, antibodies, or any other molecules that decreaseor reduce the effect of a GPCR polypeptide. Antagonists typically,diminish, inhibit, or reduce the function or activity of a GPCRmolecule.

[0148] The terms “complementary” or “complementarity” refer to thenatural binding of polynucleotides under permissive salt and temperatureconditions by base pairing. For example, the sequence “A-G-T” binds tothe complementary sequence “T-C-A”. Complementarity between twosingle-stranded molecules may be “partial”, in which only some of thenucleic acids bind, or it may be “complete” when total complementarityexists between single stranded molecules. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, which depend uponbinding between nucleic acids strands, as well as in the design and useof PNA molecules.

[0149] The term “homology” refers to a degree of complementarity. Theremay be partial homology or complete homology, wherein complete homologyis equivalent to identity. A partially complementary sequence that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid is referred to as the functional term “substantiallyhomologous”. The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (for example, Southern or Northern blot, solutionhybridization, and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. Nonetheless, conditions of low stringency do not permitnon-specific binding; low stringency conditions require that the bindingof two sequences to one another be a specific (i.e., selective)interaction. The absence of non-specific binding may be tested by theuse of a second target sequence which lacks even a partial degree ofcomplementarity (for example, less than about 30% identity). In theabsence of non-specific binding, the probe will not hybridize to thesecond non-complementary target sequence.

[0150] As used herein the terms “modulate” or “modulates” refer to anincrease or decrease in the amount, quality or effect of a particularactivity, DNA, RNA, or protein. The definition of “modulate” or“modulates” as used herein is meant to encompass agonists and/orantagonists of a particular activity, DNA, RNA, or protein.

[0151] It is another aspect of the present invention to providemodulators of the Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and/or 13protein and Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and/or 13 peptidetargets which can affect the function or activity of Gene 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 and/or 13in a cell in which Gene 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 and/or 13 function or activity is to be modulatedor affected. In addition, modulators of Gene 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 and/or 13 can affect downstream systems and molecules thatare regulated by, or which interact with, Gene 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12 and/or 13 in the cell. Modulators of Gene 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 and/or 13 include compounds, materials, agents,drugs, and the like, that antagonize, inhibit, reduce, block, suppress,diminish, decrease, or eliminate Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12 and/or 13 function and/or activity. Such compounds, materials,agents, drugs and the like can be collectively termed “antagonists”.Alternatively, modulators of Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12and/or 13 include compounds, materials, agents, drugs, and the like,that agonize, enhance, increase, augment, or amplify Gene 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 and/or 13 function in a cell. Such compounds,materials, agents, drugs and the like can be collectively termed“agonists”.

[0152] As a practical matter, whether any particular nucleic acidmolecule or polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, or 99.9% identical to a nucleotide sequence of the presentinvention can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the CLUSTALW computer program (Thompson, J. D., etal., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based onthe algorithm of Higgins, D. G., et al., Computer Applications in theBiosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment thequery and subject sequences are both DNA sequences. An RNA sequence canbe compared by converting U's to T's. However, the CLUSTALW algorithmautomatically converts U's to T's when comparing RNA sequences to DNAsequences. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a CLUSTALW alignment of DNAsequences to calculate percent identity via pairwise alignments are:Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, GapOpen Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent,Window Size=5 or the length of the subject nucleotide sequence,whichever is shorter. For multiple alignments, the following CLUSTALWparameters are preferred: Gap Opening Penalty=10; Gap ExtensionParameter=0.05; Gap Separation Penalty Range=8; End Gap SeparationPenalty=Off; % Identity for Alignment Delay=40%; Residue SpecificGaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. Thepairwise and multple alignment parameters provided for CLUSTALW aboverepresent the default parameters as provided with the AlignX softwareprogram (Vector NTI suite of programs, version 6.0).

[0153] The present invention encompasses the application of a manualcorrection to the percent identity results, in the instance where thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions. If only the local pairwisepercent identity is required, no manual correction is needed. However, amanual correction may be applied to determine the global percentidentity from a global polynucleotide alignment. Percent identitycalculations based upon global polynucleotide alignments are oftenpreferred since they reflect the percent identity between thepolynucleotide molecules as a whole (i.e., including any polynucleotideoverhangs, not just overlapping regions), as opposed to, only localmatching polynucleotides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor 5′ and 3′ truncations of the subject sequence when calculatingpercent identity. For subject sequences truncated at the 5′ or 3′ ends,relative to the query sequence, the percent identity is corrected bycalculating the number of bases of the query sequence that are 5′ and 3′of the subject sequence, which are not matched/aligned, as a percent ofthe total bases of the query sequence. Whether a nucleotide ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This corrected score may beused for the purposes of the present invention. Only bases outside the5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALWalignment, which are not matched/aligned with the query sequence, arecalculated for the purposes of manually adjusting the percent identityscore.

[0154] For example, a 90 base subject sequence is aligned to a 100 basequery sequence to determine percent identity. The deletions occur at the5′ end of the subject sequence and therefore, the CLUSTALW alignmentdoes not show a matched/alignment of the first 10 bases at 5′ end. The10 unpaired bases represent 10% of the sequence (number of bases at the5′ and 3′ ends not matched/total number of bases in the query sequence)so 10% is subtracted from the percent identity score calculated by theCLUSTALW program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by CLUSTALW is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are required for thepurposes of the present invention.

[0155] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a query amino acid sequence of the presentinvention, it is intended that the amino acid sequence of the subjectpolypeptide is identical to the query sequence except that the subjectpolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a query amino acid sequence, up to 5% of the amino acidresidues in the subject sequence may be inserted, deleted, orsubstituted with another amino acid. These alterations of the referencesequence may occur at the amino- or carboxy-terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0156] As a practical matter, whether any particular polypeptide is atleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to, for instance, an amino acid sequence referenced in Table 1(SEQ ID NO:14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) or tothe amino acid sequence encoded by cDNA contained in a deposited clone,can be determined conventionally using known computer programs. Apreferred method for determining the best overall match between a querysequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, can be determined usingthe CLUSTALW computer program (Thompson, J. D., et al., Nucleic AcidsResearch, 2(22):4673-4680, (1994)), which is based on the algorithm ofHiggins, D. G., et al., Computer Applications in the Biosciences(CABIOS), 8(2):189-191, (1992). In a sequence alignment the query andsubject sequences are both amino acid sequences. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a CLUSTALW alignment of DNA sequences to calculate percentidentity via pairwise alignments are: Matrix=BLOSUM, k-tuple=1, Numberof Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap ExtensionPenalty=0.1, Scoring Method=Percent, Window Size=5 or the length of thesubject nucleotide sequence, whichever is shorter. For multiplealignments, the following CLUSTALW parameters are preferred: Gap OpeningPenalty=10; Gap Extension Parameter=0.05; Gap Separation PenaltyRange=8; End Gap Separation Penalty=Off; % Identity for AlignmentDelay=40%;

[0157] Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; andTransition Weighting=0. The pairwise and multple alignment parametersprovided for CLUSTALW above represent the default parameters as providedwith the AlignX software program (Vector NTI suite of programs, version6.0).

[0158] The present invention encompasses the application of a manualcorrection to the percent identity results, in the instance where thesubject sequence is shorter than the query sequence because of N- orC-terminal deletions, not because of internal deletions. If only thelocal pairwise percent identity is required, no manual correction isneeded. However, a manual correction may be applied to determine theglobal percent identity from a global polypeptide alignment. Percentidentity calculations based upon global polypeptide alignments are oftenpreferred since they reflect the percent identity between thepolypeptide molecules as a whole (i.e., including any polypeptideoverhangs, not just overlapping regions), as opposed to, only localmatching polypeptides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor N- and C-terminal truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the N-and C-termini, relative to the query sequence, the percent identity iscorrected by calculating the number of residues of the query sequencethat are N- and C-terminal of the subject sequence, which are notmatched/aligned with a corresponding subject residue, as a percent ofthe total bases of the query sequence. Whether a residue ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This final percent identityscore is what may be used for the purposes of the present invention.Only residues to the N- and C-termini of the subject sequence, which arenot matched/aligned with the query sequence, are considered for thepurposes of manually adjusting the percent identity score. That is, onlyquery residue positions outside the farthest N- and C-terminal residuesof the subject sequence.

[0159] For example, a 90 amino acid residue subject sequence is alignedwith a 100 residue query sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the CLUSTALW alignment does not show a matching/alignment of the first10 residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the CLUSTALWprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence, which are not matched/aligned withthe query. In this case the percent identity calculated by CLUSTALW isnot manually corrected. Once again, only residue positions outside theN- and C-terminal ends of the subject sequence, as displayed in theCLUSTALW alignment, which are not matched/aligned with the querysequence are manually corrected for. No other manual corrections arerequired for the purposes of the present invention.

[0160] In addition to the above method of aligning two or morepolynucleotide or polypeptide sequences to arrive at a percent identityvalue for the aligned sequences, it may be desirable in somecircumstances to use a modified version of the CLUSTALW algorithm whichtakes into account known structural features of the sequences to bealigned, such as for example, the SWISS-PROT designations for eachsequence. The result of such a modifed CLUSTALW algorithm may provide amore accurate value of the percent identity for two polynucleotide orpolypeptide sequences. Support for such a modified version of CLUSTALWis provided within the CLUSTALW algorithm and would be readilyappreciated to one of skill in the art of bioinformatics.

[0161] Also available to those having skill in this art are the BLASTand BLAST 2.0 algorithms (Altschul et al., 1977, Nuc. Acids Res.,25:3389-3402 and Altschul et al., 1990, J. Mol. Biol., 215:403-410). TheBLASTN program for nucleic acid sequences uses as defaults a wordlength(W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoringmatrix (Henikoff & Henikoff, 1989, Proc. Natl. Acad. Sci., USA,89:10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4,and a comparison of both strands.

[0162] The invention encompasses polypeptides having a lower degree ofidentity but having sufficient similarity so as to perform one or moreof the same functions performed by the polypeptide of the presentinvention. Similarity is determined by conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a polypeptide by another amino acid of like characteristics(e.g., chemical properties). According to Cunningham et al above, suchconservative substitutions are likely to be phenotypically silent.Additional guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

[0163] Tolerated conservative amino acid substitutions of the presentinvention involve replacement of the aliphatic or hydrophobic aminoacids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Serand Thr; replacement of the acidic residues Asp and Glu; replacement ofthe amide residues Asn and Gln, replacement of the basic residues Lys,Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp,and replacement of the small-sized amino acids Ala, Ser, Thr, Met, andGly.

[0164] In addition, the present invention also encompasses theconservative substitutions provided in Table 4 below. TABLE 4 For AminoAcid Code Replace with any of: Alanine A D-Ala, Gly, beta-Ala, L-Cys,D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile,D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu,Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-GlnCysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln,Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp,Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala, AcpIsoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu,Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg,Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile,D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa,His, D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or5-phenylproline Proline P D-Pro, L-1-thioazolidine-4-carboxylic acid, D-or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr,Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val TyrosineY D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,D-Ile, Met, D-Met

[0165] Aside from the uses described above, such amino acidsubstitutions may also increase protein or peptide stability. Theinvention encompasses amino acid substitutions that contain, forexample, one or more non-peptide bonds (which replace the peptide bonds)in the protein or peptide sequence. Also included are substitutions thatinclude amino acid residues other than naturally occurring L-aminoacids, e.g., D-amino acids or non-naturally occurring or synthetic aminoacids, e.g., β or γ amino acids.

[0166] Both identity and similarity can be readily calculated byreference to the following publications: Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Informatics Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0167] In addition, the present invention also encompasses substitutionof amino acids based upon the probability of an amino acid substitutionresulting in conservation of function. Such probabilities are determinedby aligning multiple genes with related function and assessing therelative penalty of each substitution to proper gene function. Suchprobabilities are often described in a matrix and are used by somealgorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percentsimilarity wherein similarity refers to the degree by which one aminoacid may substitute for another amino acid without lose of function. Anexample of such a matrix is the PAM250 or BLOSUM62 matrix.

[0168] Aside from the canonical chemically conservative substitutionsreferenced above, the invention also encompasses substitutions which aretypically not classified as conservative, but that may be chemicallyconservative under certain circumstances. Analysis of enzymaticcatalysis for proteases, for example, has shown that certain amino acidswithin the active site of some enzymes may have highly perturbed pKa'sdue to the unique microenvironment of the active site. Such perturbedpKa's could enable some amino acids to substitute for other amino acidswhile conserving enzymatic structure and function. Examples of aminoacids that are known to have amino acids with perturbed pKa's are theGlu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, theHis-159 residue of Papain, etc. The conservation of function relates toeither anomalous protonation or anomalous deprotonation of such aminoacids, relative to their canonical, non-perturbed pKa. The pKaperturbation may enable these amino acids to actively participate ingeneral acid-base catalysis due to the unique ionization environmentwithin the enzyme active site. Thus, substituting an amino acid capableof serving as either a general acid or general base within themicroenvironment of an enzyme active site or cavity, as may be the case,in the same or similar capacity as the wild-type amino acid, wouldeffectively serve as a conservative amino substitution.

[0169] The present invention is directed to polynucleotide fragments ofthe polynucleotides of the invention, in addition to polypeptidesencoded therein by said polynucleotides and/or fragments.

[0170] In the present invention, a “polynucleotide fragment” refers to ashort polynucleotide having a nucleic acid sequence which: is a portionof that contained in a deposited clone, or encoding the polypeptideencoded by the cDNA in a deposited clone; is a portion of that shown inSEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 or thecomplementary strand thereto, or is a portion of a polynucleotidesequence encoding the polypeptide of SEQ ID NO:14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, or 26. The nucleotide fragments of the inventionare preferably at least about 15 nt, and more preferably at least about20 nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt, at least about 50 nt, at least about75 nt, or at least about 150 nt in length. A fragment “at least 20 nt inlength,” for example, is intended to include 20 or more contiguous basesfrom the cDNA sequence contained in a deposited clone or the nucleotidesequence shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or13. In this context “about” includes the particularly recited value, avalue larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, ateither terminus, or at both termini. These nucleotide fragments haveuses that include, but are not limited to, as diagnostic probes andprimers as discussed herein. Of course, larger fragments (e.g., 50, 150,500, 600, 2000 nucleotides) are preferred.

[0171] Moreover, representative examples of polynucleotide fragments ofthe invention, include, for example, fragments comprising, oralternatively consisting of, a sequence from about nucleotide number1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400,401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850,851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200,1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ IDNO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, or the complementarystrand thereto, or the cDNA contained in a deposited clone. In thiscontext “about” includes the particularly recited ranges, and rangeslarger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini. Preferably, these fragments encode apolypeptide which has biological activity. More preferably, thesepolynucleotides can be used as probes or primers as discussed herein.Also encompassed by the present invention are polynucleotides whichhybridize to these nucleic acid molecules under stringent hybridizationconditions or lower stringency conditions, as are the polypeptidesencoded by these polynucleotides.

[0172] In the present invention, a “polypeptide fragment” refers to anamino acid sequence which is a portion of that contained in SEQ IDNO:14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 or encoded bythe cDNA contained in a deposited clone. Protein (polypeptide) fragmentsmay be “free-standing,” or comprised within a larger polypeptide ofwhich the fragment forms a part or region, most preferably as a singlecontinuous region. Representative examples of polypeptide fragments ofthe invention, include, for example, fragments comprising, oralternatively consisting of, from about amino acid number 1-20, 21-40,41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end ofthe coding region. Moreover, polypeptide fragments can be about 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids inlength. In this context “about” includes the particularly recited rangesor values, and ranges or values larger or smaller by several (5, 4, 3,2, or 1) amino acids, at either extreme or at both extremes.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0173] Preferred polypeptide fragments include the full-length protein.Further preferred polypeptide fragments include the full-length proteinhaving a continuous series of deleted residues from the amino or thecarboxy terminus, or both. For example, any number of amino acids,ranging from 1-60, can be deleted from the amino terminus of thefull-length polypeptide. Similarly, any number of amino acids, rangingfrom 1-30, can be deleted from the carboxy terminus of the full-lengthprotein. Furthermore, any combination of the above amino and carboxyterminus deletions are preferred. Similarly, polynucleotides encodingthese polypeptide fragments are also preferred.

[0174] Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, or 26 falling within conserved domains arespecifically contemplated by the present invention. Moreover,polynucleotides encoding these domains are also contemplated.

[0175] Other preferred polypeptide fragments are biologically activefragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of thepolypeptide of the present invention. The biological activity of thefragments may include an improved desired activity, or a decreasedundesirable activity. Polynucleotides encoding these polypeptidefragments are also encompassed by the invention.

[0176] In a preferred embodiment, the functional activity displayed by apolypeptide encoded by a polynucleotide fragment of the invention may beone or more biological activities typically associated with thefull-length polypeptide of the invention. Illustrative of thesebiological activities includes the fragments ability to bind to at leastone of the same antibodies which bind to the full-length protein, thefragments ability to interact with at lease one of the same proteinswhich bind to the full-length, the fragments ability to elicit at leastone of the same immune responses as the full-length protein (i.e., tocause the immune system to create antibodies specific to the sameepitope, etc.), the fragments ability to bind to at least one of thesame polynucleotides as the full-length protein, the fragments abilityto bind to a receptor of the full-length protein, the fragments abilityto bind to a ligand of the full-length protein, and the fragmentsability to multimerize with the full-length protein. However, theskilled artisan would appreciate that some fragments may have biologicalactivities which are desirable and directly inapposite to the biologicalactivity of the full-length protein. The functional activity ofpolypeptides of the invention, including fragments, variants,derivatives, and analogs thereof can be determined by numerous methodsavailable to the skilled artisan, some of which are described elsewhereherein.

[0177] The term “hybridization” refers to any process by which a strandof nucleic acids binds with a complementary strand through base pairing.The term “hybridization complex” refers to a complex formed between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary G and C bases and between complementary A and Tbases. The hydrogen bonds may be further stabilized by base stackinginteractions. The two complementary nucleic acid sequences hydrogen bondin an anti-parallel configuration. A hybridization complex may be formedin solution (for example, C_(o)t or R_(o)t analysis), or between onenucleic acid sequence present in solution and another nucleic acidsequence immobilized on a solid phase or support (for example,membranes, filters, chips, pins, or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenaffixed).

[0178] The terms “stringency” or “stringent conditions” refer to theconditions for hybridization as defined by nucleic acid composition,salt, and temperature. These conditions are well known in the art andmay be altered to identify and/or detect identical or relatedpolynucleotide sequences in a sample. A variety of equivalent conditionscomprising either low, moderate, or high stringency depend on factorssuch as the length and nature of the sequence (DNA, RNA, basecomposition), reaction milieu (in solution or immobilized on a solidsubstrate), nature of the target nucleic acid (DNA, RNA, basecomposition), concentration of salts and the presence or absence ofother reaction components (for example, formamide, dextran sulfateand/or polyethylene glycol) and reaction temperature (within a range offrom about 5° C. below the melting temperature of the probe to about 20°C. to 25° C. below the melting temperature). One or more factors may bevaried to generate conditions, either low or high stringency that isdifferent from but equivalent to the aforementioned conditions.

[0179] As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences. As will be furtherappreciated by the skilled practitioner, the melting temperature, Tm,can be approximated by the formulas as well known in the art, dependingon a number of parameters, such as the length of the hybrid or probe innumber of nucleotides, or hybridization buffer ingredients andconditions (see, for example, T. Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1982 and J. Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Current Protocols in Molecular Biology, Eds. F. M. Ausubel et al., Vol.1, “Preparation and Analysis of DNA”, John Wiley and Sons, Inc.,1994-1995, Suppls. 26, 29, 35 and 42;

[0180] pp. 2.10.7-2.10.16; G. M. Wahl and S. L. Berger (1987; MethodsEnzymol. 152:399-407); and A. R. Kimmel, 1987; Methods of Enzymol.152:507-511).

[0181] As a general guide, T_(m) decreases approximately 1° C.-1.5° C.with every 1% decrease in sequence homology. Also, in general, thestability of a hybrid is a function of sodium ion concentration andtemperature. Typically, the hybridization reaction is initiallyperformed under conditions of low stringency, followed by washes ofvarying, but higher stringency. Reference to hybridization stringency,for example, high, moderate, or low stringency, typically relates tosuch washing conditions. It is to be understood that the low, moderateand high stringency hybridization or washing conditions can be variedusing a variety of ingredients, buffers and temperatures well known toand practiced by the skilled artisan.

[0182] A “composition”, as defined herein, refers broadly to anycomposition containing a GPCR polynucleotide, polypeptide, derivative,or mimetic thereof, or antibodies thereto. The composition may comprisea dry formulation or an aqueous solution. Compositions comprising GPCRpolynucleotide sequences (SEQ ID NOs:1-13) encoding GPCR polypeptides(SEQ ID NOs:14-26), or fragments thereof, may be employed ashybridization probes. The probes may be stored in a freeze-dried formand may be in association with a stabilizing agent such as acarbohydrate. In hybridizations, the probe may be employed in an aqueoussolution containing salts (for example, NaCl), detergents or surfactants(for example, SDS) and other components (for example, Denhardt'ssolution, dry milk, salmon sperm DNA, and the like).

[0183] The term “substantially purified” refers to nucleic acidsequences or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% to 85% free, and most preferably 90% to 95%, or greater,free from other components with which they are naturally associated.

[0184] The term “sample”, or “biological sample”, is meant to beinterpreted in its broadest sense. A non-limiting example of abiological sample suspected of containing a GPCR nucleic acid encodingGPCR protein, or fragments thereof, or a GPCR protein itself, maycomprise, but is not limited to, a body fluid, an extract from cells ortissue, chromosomes isolated from a cell (for example, a spread ofmetaphase chromosomes), organelle, or membrane isolated from a cell, acell, nucleic acid such as genomic GPCR DNA (in solution or bound to asolid support such as, for example, for Southern analysis), GPCR RNA (insolution or bound to a solid support such as for Northern analysis),GPCR cDNA (in solution or bound to a solid support), a tissue, a tissueprint, and the like.

[0185] “Transformation” or transfection refers to a process by whichexogenous DNA, preferably GPCR, enters and changes a recipient cell. Itmay occur under natural or artificial conditions using various methodswell known in the art. Transformation may rely on any known method forthe insertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method is selected based on the type of hostcell being transformed and may include, but is not limited to, viralinfection, electroporation, heat shock, lipofection, and partialbombardment. Such “transformed” cells include stably transformed cellsin which the inserted DNA is capable of replication either as anautonomously replicating plasmid or as part of the host chromosome.Transformed cells also include those cells, which transiently expressthe inserted DNA or RNA for limited periods of time.

[0186] The term “correlates with expression of a polynucleotide”indicates that the detection of the presence of ribonucleic acid that issimilar to the nucleic acid sequence of GPCRs by Northern analysis isindicative of the presence of mRNA encoding GPCR polypeptides (SEQ IDNOs:14-26) in a sample and thereby correlates with expression of thetranscript from the polynucleotide encoding the protein.

[0187] An alteration in the polynucleotide of SEQ ID NOs:1-13 comprisesany alteration in the sequence of the polynucleotides encoding GPCRpolypeptides, including deletions, insertions, and point mutations thatmay be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes GPCR polypeptides (e.g., by alterations in the pattern ofrestriction fragment length polymorphisms capable of hybridizing tonucleic acid sequences SEQ ID NOs:1-13), the inability of a selectedfragment of SEQ ID NOs:1-13 to hybridize to a sample of genomic DNA(e.g., using allele-specific oligonucleotide probes), and improper orunexpected hybridization, such as hybridization to a locus other thanthe normal chromosomal locus for the polynucleotide sequence encodingGPCR polypeptide (e.g., using fluorescent in situ hybridization (FISH)to metaphase chromosome spreads).

[0188] The term “antibody” refers to intact molecules as well asfragments thereof, such as Fab, F(ab′)₂, Fv, which are capable ofbinding an epitopic or antigenic determinant. Antibodies that bind toGPCR polypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest or prepared recombinantly for useas the immunizing antigen. The polypeptide or oligopeptide used toimmunize an animal can be derived from the transition of RNA orsynthesized chemically, and can be conjugated to a carrier protein, ifdesired. Commonly used carriers that are chemically coupled to peptidesinclude, but are not limited to, bovine serum albumin (BSA), keyholelimpet hemocyanin (KLH), and thyroglobulin. The coupled peptide is thenused to immunize the animal (for example, a mouse, a rat, or a rabbit).

[0189] The term “humanized” antibody refers to antibody molecules inwhich amino acids have been replaced in the non-antigen binding regions(i.e., framework regions) of the immunoglobulin in order to more closelyresemble a human antibody, while still retaining the original bindingcapability, for example, as described in U.S. Pat. No. 5,585,089 to C.L. Queen et al. In the present instance, humanized antibodies arepreferably anti-GPCR specific antibodies.

[0190] The term “antigenic determinant” refers to that portion of amolecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein, preferably a GPCRprotein, is used to immunize a host animal, numerous regions of theprotein may induce the production of antibodies which bind specificallyto a given region or three-dimensional structure on the protein; theseregions or structures are referred to an antigenic determinants. Anantigenic determinant may compete with the intact antigen (i.e., theimmunogen used to elicit the immune response) for binding to anantibody.

[0191] The terms “specific binding” or “specifically binding” refer tothe interaction between a protein or peptide, preferably a GPCR protein,and a binding molecule, such as an agonist, an antagonist, or anantibody. The interaction is dependent upon the presence of a particularstructure (i.e., an antigenic determinant or epitope) of the proteinthat is recognized by the binding molecule.

[0192] The present invention provides novel GPCR polynucleotides andencoded GPCR polypeptides. The GPCRs according to this invention arepreferably full-length molecules. More specifically, the GPCRs accordingto the invention are sensory GPCRs, chemokine GPCRs, orphan GPCRs, andvery large GPCRs.

[0193] GPCRs can also include dopamine receptors, rhodopsin receptors,kinin receptors, N-formyl peptide receptors, opioid receptors,calcitonin receptors, adrenergic receptors, endothelin receptors, cAMPreceptors, adenosine receptors, muscarinic receptors, acetylcholinereceptors, serotonin receptors, histamine receptors, thrombin receptors,follicle stimulating hormone receptors, opsin receptors, endothelialdifferentiation gene-1 receptors, odorant receptors, or cytomegalovirusreceptors.

[0194] Features of the Polypeptide Encoded by Gene No:7

[0195] The determined nucleotide sequence of the Gene 7 cDNA in FIGS.13A-C (SEQ ID NO:7) contains an open reading frame encoding a protein ofabout 328 amino acid residues, with a deduced molecular weight of about36.9 kDa. The amino acid sequence of the predicted Gene 7 polypeptide isshown in FIGS. 13A-C and in FIG. 14 (SEQ ID NO:20).

[0196] The Gene 7 polypeptide was predicted to comprise seventransmembrane domains (TM1 to TM7) located from about amino acid 28 toabout amino acid 53 (TM1; SEQ ID NO:87); from about amino acid 63 toabout amino acid 88 (TM2; SEQ ID NO:88); from about amino acid 101 toabout amino acid 122 (TM3; SEQ ID NO:89); from about amino acid 142 toabout amino acid 160 (TM4; SEQ ID NO:90); from about amino acid 182 toabout amino acid 204 (TM5; SEQ ID NO:91); from about amino acid 218 toabout amino acid 238 (TM6; SEQ ID NO:92); and/or from about amino acid260 to about amino acid 279 (TM7; SEQ ID NO:93) of SEQ ID NO:20 (FIGS.13A-C and FIG. 14). In this context, the term “about” may be construedto mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond theN-Terminus and/or C-terminus of the above referenced transmembranedomain polypeptides.

[0197] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention:LSFTGLTCIVSLVALTGNAVVLWLLG (SEQ ID NO:87), IYILNLVAADFLFLCFQIINCLVYLS(SEQ ID NO:88), FFTTVMTCAYLAGLSMLSTVST (SEQ ID NO:89),LSAVVCVLLWALSLLLSIL (SEQ ID NO:90), FITAAWLIFLFMVLCGSSLALLV (SEQ IDNO:91), LYLTILLTVLVFLLCGLPFGI (SEQ ID NO:92), and/orVSVVLSSLNSSANPIIYFFV (SEQ ID NO:93). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these Gene 7 transmembrane domain polypeptides as immunogenicand/or antigenic epitopes as described elsewhere herein.

[0198] In preferred embodiments, the present invention encompasses theuse of N-terminal deletions, C-terminal deletions, or any combination ofN-terminal and C-terminal deletions of any one or more of the Gene 7 TM1thru TM7 transmembrane domain polypeptides as antigenic and/orimmunogenic epitopes.

[0199] In preferred embodiments, the present invention also encompassesthe use of N-terminal deletions, C-terminal deletions, or anycombination of N-terminal and C-terminal deletions of any one or more ofthe amino acids intervening (i.e., GPCR extracellular or intracellularloops) any pair of Gene 7 TM1 thru TM7 transmembrane domainpolypeptides, and/or the amino acids intervening any pair of the Gene 7TM1 thru TM7 transmembrane domain polypeptides themselves, as antigenicand/or immunogenic epitopes.

[0200] The coding region of the Gene 7 polynucleotide is predicted to befrom nucleotide 169 to nucleotide 1152 of SEQ ID NO:7 as shown in FIGS.13A-C, and the polypeptide corresponding to amino acids 1 thru 328 ofSEQ ID NO:20. The present invention encompasses the polynucleotideencompassing the entire coding region of Gene 7.

[0201] Alternatively, the present invention encompasses a polynucleotidelacking the initiating start codon, in addition to, the resultingencoded polypeptide of Gene 7. Specifically, the present inventionencompasses the polynucleotide corresponding to nucleotides 172 thru1152 of SEQ ID NO:7, and the polypeptide corresponding to amino acids 2thru 328 of SEQ ID NO:20. Also encompassed are recombinant vectorscomprising said encoding sequence, and host cells comprising saidvector.

[0202] In preferred embodiments, the following N-terminal Gene 7deletion polypeptides are encompassed by the present invention: M1-Q328,D2-Q328, S3-Q328, T4-Q328, I5-Q328, P6-Q328, V7-Q328, L8-Q328, G9-Q328,T10-Q328, E11-Q328, L12-Q328, T13-Q328, P14-Q328, I15-Q328, N16-Q328,G17-Q328, R18-Q328, E19-Q328, E20-Q328, T21-Q328, P22-Q328, C23-Q328,Y24-Q328, K25-Q328, Q26-Q328, T27-Q328, L28-Q328, S29-Q328, F30-Q328,T31-Q328, G32-Q328, L33-Q328, T34-Q328, C35-Q328, 136-Q328, V37-Q328,S38-Q328, L39-Q328, V40-Q328, A41-Q328, L42-Q328, T43-Q328, G44-Q328,N45-Q328, A46-Q328, V47-Q328, V48-Q328, L49-Q328, W50-Q328, L51-Q328,L52-Q328, G53-Q328, C54-Q328, R55-Q328, M56-Q328, R57-Q328, R58-Q328,N59-Q328, A60-Q328, V61-Q328, S62-Q328, I63-Q328, Y64-Q328, I65-Q328,L66-Q328, N67-Q328, L68-Q328, V69-Q328, A70-Q328, A71-Q328, D72-Q328,F73-Q328, L74-Q328, F75-Q328, L76-Q328, C77-Q328, F78-Q328, Q79-Q328,I80-Q328, I81-Q328, N82-Q328, C83-Q328, L84-Q328, V85-Q328, Y86-Q328,L87-Q328, S88-Q328, N89-Q328, F90-Q328, F91-Q328, C92-Q328, S93-Q328,I94-Q328, S95-Q328, I96-Q328, N97-Q328, F98-Q328, P99-Q328, S100-Q328,F101-Q328, F102-Q328, T103-Q328, T104-Q328, V105-Q328, M106-Q328,T107-Q328, C108-Q328, A109-Q328, Y100-Q328, L111-Q328, A112-Q328,G113-Q328, L114-Q328, S115-Q328, M116-Q328, L117-Q328, S118-Q328,T119-Q328, V120-Q328, S121-Q328, T122-Q328, E123-Q328, R124-Q328,C125-Q328, L126-Q328, S127-Q328, V128-Q328, L129-Q328, W130-Q328,P131-Q328, I132-Q328, W133-Q328, Y134-Q328, R135-Q328, C136-Q328,R137-Q328, R138-Q328, P139-Q328, R140-Q328, H141-Q328, L142-Q328,S143-Q328, A144-Q328, V145-Q328, V146-Q328, C147-Q328, V148-Q328,L149-Q328, L150-Q328, W151-Q328, A152-Q328, L153-Q328, S154-Q328,L155-Q328, L156-Q328, L157-Q328, S158-Q328, 1159-Q328, L160-Q328,E161-Q328, G162-Q328, K163-Q328, F164-Q328, C165-Q328, G166-Q328,F167-Q328, L168-Q328, F169-Q328, S170-Q328, D171-Q328, G172-Q328,D173-Q328, S174-Q328, G175-Q328, W176-Q328, C177-Q328, Q178-Q328,T179-Q328, F180-Q328, D181-Q328, F182-Q328, I183-Q328, T184-Q328,A185-Q328, A186-Q328, W187-Q328, L188-Q328, I189-Q328, F190-Q328,L191-Q328, F192-Q328, M193-Q328, V194-Q328, L195-Q328, C196-Q328,G197-Q328, S198-Q328, S199-Q328, L200-Q328, A201-Q328, L202-Q328,L203-Q328, V204-Q328, R205-Q328, I206-Q328, L207-Q328, C208-Q328,G209-Q328, S210-Q328, R211-Q328, G212-Q328, L213-Q328, P214-Q328,L215-Q328, T216-Q328, R217-Q328, L218-Q328, Y219-Q328, L220-Q328,T221-Q328, I222-Q328, L223-Q328, L224-Q328, T225-Q328, V226-Q328,L227-Q328, V228-Q328, F229-Q328, L230-Q328, L231-Q328, C232-Q328,G233-Q328, L234-Q328, P235-Q328, F236-Q328, G237-Q328, I238-Q328,Q239-Q328, W240-Q328, F241-Q328, L242-Q328, I243-Q328, L244-Q328,W245-Q328, I246-Q328, W247-Q328, K248-Q328, D249-Q328, S250-Q328,D251-Q328, V252-Q328, L253-Q328, F254-Q328, C255-Q328, H256-Q328,I257-Q328, H258-Q328, P259-Q328, V260-Q328, S261-Q328, V262-Q328,V263-Q328, L264-Q328, S265-Q328, S266-Q328, L267-Q328, N268-Q328,S269-Q328, S270-Q328, A271-Q328, N272-Q328, P273-Q328, I274-Q328,1275-Q328, Y276-Q328, F277-Q328, F278-Q328, V279-Q328, G280-Q328,S281-Q328, F282-Q328, R283-Q328, K284-Q328, Q285-Q328, W286-Q328,R287-Q328, L288-Q328, Q289-Q328, Q290-Q328, P291-Q328, I292-Q328,L293-Q328, K294-Q328, L295-Q328, A296-Q328, L297-Q328, Q298-Q328,R299-Q328, A300-Q328, L301-Q328, Q302-Q328, D303-Q328, I304-Q328,A305-Q328, E306-Q328, V307-Q328, D308-Q328, E309-Q328, G310-Q328,G311-Q328, G312-Q328, W313-Q328, L314-Q328, P315-Q328, Q316-Q328,E317-Q328, T318-Q328, L319-Q328, E320-Q328, L321-Q328, and/or S322-Q328of SEQ ID NO:20. Polynucleotide sequences encoding these polypeptidesare also provided. The present invention also encompasses the use ofthese N-terminal Gene 7 deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

[0203] In preferred embodiments, the following C-terminal Gene 7deletion polypeptides are encompassed by the present invention: M1-Q328,M1-E327, M1-L326, M1-R325, M1-S324, M1-G323, M1-S322, M1-L321, M1-E320,M1-L319, M1-T318, M1-E317, M1-Q316, M1-P315, M1-L314, M1-W313, M1-G312,M1-G311, M1-G310, M1-E309, M1-D308, M1-V307, M1-E306, M1-A305, M1-I304,M1-D303, M1-Q302, M1-L301, M1-A300, M1-R299, M1-Q298, M1-L297, M1-A296,M1-L295, M1-K294, M1-L293, M1-I292, M1-P291, M1-Q290, M1-Q289, M1-L288,M1-R287, M1-W286, M1-Q285, M1-K284, M1-R283, M1-F282, M1-S281, M1-G280,M1-V279, M1-F278, M1-F277, M1-Y276, M1-I275, M1-I274, M1-P273, M1-N272,M1-A271, M1-S270, M1-S269, M1-N268, M1-L267, M1-S266, M1-S265, M1-L264,M1-V263, M1-V262, M1-S261, M1-V260, M1-P259, M1-H258, M1-I257, M1-H256,M1-C255, M1-F254, M1-L253, M1-V252, M1-D251, M1-S250, M1-D249, M1-K248,M1-W247, M1-I246, M1-W245, M1-L244, M1-I243, M1-L242, M1-F241, M1-W240,M1-Q239, M1-I238, M1-G237, M1-F236, M1-P235, M1-L234, M1-G233, M1-C232,M1-L231, M1-L230, M1-F229, M1-V228, M1-L227, M1-V226, M1-T225, M1-L224,M1-L223, M1-I222, M1-T221, M1-L220, M1-Y219, M1-L218, M1-R217, M1-T216,M1-L215, M1-P214, M1-L213, M1-G212, M1-R211, M1-S210, M1-G209, M1-C208,M1-L207, M1-I206, M1-R205, M1-V204, M1-L203, M1-L202, M1-A201, M1-L200,M1-S199, M1-S198, M1-G197, M1-C196, M1-L195, M1-V194, M1-M193, M1-F192,M1-L191, M1-F190, M1-I189, M1-L188, M1-W187, M1-A186, M1-A185, M1-T184,M1-I183, M1-F182, M1-D181, M1-F180, M1-T179, M1-Q178, M1-C₁₇₇, M1-W176,M1-G175, M1-S174, M1-D173, M1-G172, M1-D171, M1-S170, M1-F169, M1-L168,M1-F167, M1-G166, M1-C165, M1-F164, M1-K163, M1-G162, M1-E161, M1-L160,M1-I159, M1-S158, M1-L157, M1-L156, M1-L155, M1-S154, M1-L153, M1-A152,M1-W151, M1-L150, M1-L149, M1-V148, M1-C₁₄₇, M1-V146, M1-V145, M1-A144,M1-S143, M1-L142, M1-H141, M1-R140, M1-P139, M1-R138, M1-R137, M1-C136,M1-R135, M1-Y134, M1-W133, M1-I132, M1-P131, M1-W130, M1-L129, M1-V128,M1-S127, M1-L126, M1-C125, M1-R124, M1-E123, M1-T122, M1-S121, M1-V120,M1-T119, M1-S118, M1-L117, M1-M116, M1-S115, M1-L114, M1-G113, M1-A112,M1-L111, M1-Y110, M1-A109, M1-C108, M1-T107, M1-M106, M1-V105, M1-T104,M1-T103, M1-F102, M1-F110, M1-S100, M1-P99, M1-F98, M1-N97, M1-I96,M1-S95, M1-I94, M1-S93, M1-C92, M1-F91, M1-F90, M1-N89, M1-S88, M1-L87,M1-Y86, M1-V85, M1-L84, M1-C83, M1-N82, M1-I81, M1-I80, M1-Q79, M1-F78,M1-C77, M1-L76, M1-F75, M1-L74, M1-F73, M1-D72, M1-A71, M1-A70, M1-V69,M1-L68, M1-N67, M1-L66, M1-I65, M1-Y64, M1-I63, M1-S62, M1-V61, M1-A60,M1-N59, M1-R58, M1-R57, M1-M56, M1-R55, M1-C54, M1-G53, M1-L52, M1-L51,M1-W50, M1-L49, M1-V48, M1-V47, M1-A46, M1-N45, M1-G44, M1-T43, M1-L42,M1-A41, M1-V40, M1-L39, M1-S38, M1-V37, M1-I36, M1-C35, M1-T34, M1-L33,M1-G32, M1-T31, M1-F30, M1-S29, M1-L28, M1-T27, M1-Q26, M1-K25, M1-Y24,M1-C23, M1-P22, M1-T21, M1-E20, M1-E19, M1-R18, M1-G17, M1-N16, M1-I15,M1-P14, M1-T13, M1-L12, M1-E11, M1-T10, M1-G9, M1-L8, and/or M1-V7 ofSEQ ID NO:20. Polynucleotide sequences encoding these polypeptides arealso provided. The present invention also encompasses the use of theseC-terminal Gene 7 deletion polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0204] The Gene 7 polypeptides of the present invention were determinedto comprise several phosphorylation sites based upon the Motif algorithm(Genetics Computer Group, Inc.). The phosphorylation of such sites mayregulate some biological activity of the Gene 7 polypeptide. Forexample, phosphorylation at specific sites may be involved in regulatingthe proteins ability to associate or bind to other molecules (e.g.,proteins, ligands, substrates, DNA, etc.). In the present case,phosphorylation may modulate the ability of the Gene 7 polypeptide toassociate with other polypeptides, particularly cognate ligand for Gene7, or its ability to modulate certain cellular signal pathways.

[0205] The Gene 7 polypeptide was predicted to comprise two PKCphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem.260:12492-12499(1985); which are hereby incorporated by referenceherein.

[0206] In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: LSTVSTERCLSVL(SEQ ID NO:94), and/or YFFVGSFRKQWRL (SEQ ID NO:95). Polynucleotidesencoding these polypeptides are also provided. The present inventionalso encompasses the use of the Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or 13 PKC phosphorylation site polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein. The Gene 7 polypeptidewas predicted to comprise three casein kinase II phosphorylation sitesusing the Motif algorithm (Genetics Computer Group, Inc.). Casein kinaseII (CK-2) is a protein serine/threonine kinase whose activity isindependent of cyclic nucleotides and calcium. CK-2 phosphorylates manydifferent proteins. The substrate specificity [1] of this enzyme can besummarized as follows: (1) Under comparable conditions Ser is favoredover Thr.; (2) An acidic residue (either Asp or Glu) must be presentthree residues from the C-terminal of the phosphate acceptor site; (3)Additional acidic residues in positions +1, +2, +4, and +5 increase thephosphorylation rate. Most physiological substrates have at least oneacidic residue in these positions; (4) Asp is preferred to Glu as theprovider of acidic determinants; and (5) A basic residue at theN-terminal of the acceptor site decreases the phosphorylation rate,while an acidic one will increase it.

[0207] A consensus pattern for casein kinase II phosphorylations site isas follows: [ST]-x(2)-[DE], wherein ‘x’ represents any amino acid, and Sor T is the phosphorylation site.

[0208] Additional information specific to casein kinase IIphosphorylation sites may be found in reference to the followingpublication: Pinna L. A., Biochim. Biophys. Acta 1054:267-284(1990);which is hereby incorporated herein in its entirety.

[0209] In preferred embodiments, the following casein kinase IIphosphorylation site polypeptide is encompassed by the presentinvention: LSLLLSILEGKFCG (SEQ ID NO:96), CGFLFSDGDSGWCQ (SEQ ID NO:97),and/or LELSGSRLEQ (SEQ ID NO:98). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of this casein kinase II phosphorylation site polypeptide as animmunogenic and/or antigenic epitope as described elsewhere herein.

[0210] The Gene 7 polypeptide has been shown to comprise oneglycosylation site according to the Motif algorithm (Genetics ComputerGroup, Inc.). As discussed more specifically herein, proteinglycosylation is thought to serve a variety of functions including:augmentation of protein folding, inhibition of protein aggregation,regulation of intracellular trafficking to organelles, increasingresistance to proteolysis, modulation of protein antigenicity, andmediation of intercellular adhesion.

[0211] Asparagine glycosylation sites have the following consensuspattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site.However, it is well known that that potential N-glycosylation sites arespecific to the consensus sequence Asn-Xaa-Ser/Thr. However, thepresence of the consensus tripeptide is not sufficient to conclude thatan asparagine residue is glycosylated, due to the fact that the foldingof the protein plays an important role in the regulation ofN-glycosylation. It has been shown that the presence of proline betweenAsn and Ser/Thr will inhibit N-glycosylation; this has been confirmed bya recent statistical analysis of glycosylation sites, which also showsthat about 50% of the sites that have a proline C-terminal to Ser/Thrare not glycosylated. Additional information relating to asparagineglycosylation may be found in reference to the following publications,which are hereby incorporated by reference herein: Marshall R. D., Annu.Rev. Biochem. 41:673-702(1972); Pless D. D., Lennarz W. J., Proc. Natl.Acad. Sci. U.S.A. 74:134-138(1977); Bause E., Biochem. J.209:331-336(1983); Gavel Y., von Heijne G., Protein Eng.3:433-442(1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem.265:11397-11404(1990).

[0212] In preferred embodiments, the following asparagine glycosylationsite polypeptides are encompassed by the present invention:VLSSLNSSANPIIY (SEQ ID NO:99). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of this Gene 7 asparagine glycosylation site polypeptide as animmunogenic and/or antigenic epitope as described elsewhere herein.

[0213] The Gene 7 polypeptide was predicted to comprise threeN-myristoylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). An appreciable number of eukaryotic proteins are acylatedby the covalent addition of myristate (a C14-saturated fatty acid) totheir N-terminal residue via an amide linkage. The sequence specificityof the enzyme responsible for this modification, myristoyl CoA:proteinN-myristoyl transferase (NMT), has been derived from the sequence ofknown N-myristoylated proteins and from studies using syntheticpeptides. The specificity seems to be the following: i.) The N-terminalresidue must be glycine; ii.) In position 2, uncharged residues areallowed; iii.) Charged residues, proline and large hydrophobic residuesare not allowed; iv.) In positions 3 and 4, most, if not all, residuesare allowed; v.) In position 5, small uncharged residues are allowed(Ala, Ser, Thr, Cys, Asn and Gly). Serine is favored; and vi.) Inposition 6, proline is not allowed.

[0214] A consensus pattern for N-myristoylation is as follows:G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}, wherein ‘x’ represents any amino acid,and G is the N-myristoylation site.

[0215] Additional information specific to N-myristoylation sites may befound in reference to the following publication: Towler D. A., Gordon J.I., Adams S. P., Glaser L., Annu. Rev. Biochem. 57:69-99(1988); andGrand R. J. A., Biochem. J. 258:625-638(1989); which is herebyincorporated herein in its entirety.

[0216] In preferred embodiments, the following N-myristoylation sitepolypeptides are encompassed by the present invention: FMVLCGSSLALLVRIL(SEQ ID NO:100), LCGSRGLPLTRLYLTI (SEQ ID NO:101), and/orVFLLCGLPFGIQWFLI (SEQ ID NO:102). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these N-myristoylation site polypeptides as immunogenicand/or antigenic epitopes as described elsewhere herein.

[0217] The Gene 7 polypeptide has been shown to comprise one leucinezipper site according to the Motif algorithm (Genetics Computer Group,Inc.). Leucine zipper sites have been proposed to explain how someeukaryotic gene regulatory proteins work. The leucine zipper consists ofa periodic repetition of leucine residues at every seventh position overa distance covering eight helical turns. The segments containing theseperiodic arrays of leucine residues seem to exist in an alpha-helicalconformation. The leucine side chains extending from one alpha-helixinteract with those from a similar alpha helix of a second polypeptide,facilitating dimerization; the structure formed by cooperation of thesetwo regions forms a coiled coil. The leucine zipper pattern is presentin many gene regulatory proteins, such as i.) the CCATT-box and enhancerbinding protein (C/EBP); ii) the cAMP response element (CRE) bindingproteins (CREB, CRE-BP1, ATFs); the Jun/AP1 family of transcriptionfactors; iv.) the yeast general control protein GCN4; v.) the fosoncogene, and the fos-related proteins fra-1 and fos B; vi.) the C-myc,L-myc and N-myc oncogenes; and vii.) the octamer-binding transcriptionfactor 2 (Oct-2/OTF-2). Leucine zipper motifs have the followingconcensus pattern: L-x(6)-L-x(6)-L-x(6)-L, wherein ‘x’ represents anyamino acid. Additional information relating to leucine zipper motifs maybe found in reference to the following publications, which are herebyincorporated by reference herein: Landschulz W. H., Johnson P. F.,McKnight S. L., Science 240:1759-1764(1988); Busch S. J., Sassone-CorsiP., Trends Genet. 6:36-40(1990); and/or O'Shea E. K., Rutkowski R., KimP. S., Science 243:538-542(1989).

[0218] In preferred embodiments, the following leucine zipper sitepolypeptide is encompassed by the present invention:CGSRGLPLTRLYLTILLTVLVFLLCGLPFGIQ (SEQ ID NO:103). Polynucleotidesencoding these polypeptides are also provided. The present inventionalso encompasses the use of this Gene 7 leucine zipper site polypeptideas an immunogenic and/or antigenic epitope as described elsewhereherein.

[0219] Moreover, in confirmation of Gene 7 representing a novel GPCR,the Gene 7 polypeptide was predicted to comprise a G-protein coupledreceptor motif using the Motif algorithm (Genetics Computer Group,Inc.). G-protein coupled receptors (also called R7G) are an extensivegroup of hormones, neurotransmitters, odorants and light receptors whichtransduce extracellular signals by interaction with guaninenucleotide-binding (G) proteins. Some examples of receptors that belongto this family are provided as follows: 5-hydroxytryptamine (serotonin)1A to 1F, 2A to 2C, 4, SA, 5B, 6 and 7, Acetylcholine, muscarinic-type,M1 to M5, Adenosine A1, A2A, A2B and A3, Adrenergic alpha-1A to -1C;alpha-2A to -2D; beta-1 to -3, Angiotensin II types I and II, Bombesinsubtypes 3 and 4, Bradykinin B1 and B2, c3a and C5a anaphylatoxin,Cannabinoid CB1 and CB2, Chemokines C—C CC-CKR-1 to CC-CKR-8, ChemokinesC-X-C CXC-CKR-1 to CXC-CKR-4, Cholecystokinin-A andcholecystokinin-B/gastrin, Dopamine D1 to D5, Endothelin ET-a and ET-b,fMet-Leu-Phe (fMLP) (N-formyl peptide), Follicle stimulating hormone(FSH-R), Galanin, Gastrin-releasing peptide (GRP-R),Gonadotropin-releasing hormone (GNRH-R), Histamine H1 and H2 (gastricreceptor I), Lutropin-choriogonadotropic hormone (LSH-R), MelanocortinMC1R to MC5R, Melatonin, Neuromedin B (NMB-R), Neuromedin K (NK-3R),Neuropeptide Y types 1 to 6, Neurotensin (NT-R), Octopamine (tyramine)from insects, Odorants, Opioids delta-, kappa- and mu-types, Oxytocin(OT-R), Platelet activating factor (PAF-R), Prostacyclin, ProstaglandinD2, Prostaglandin E2, EP1 to EP4 subtypes, Prostaglandin F2,Purinoreceptors (ATP), Somatostatin types 1 to 5, Substance-K (NK-2R),Substance-P (NK-1R), Thrombin, Thromboxane A2, Thyrotropin (TSH-R),Thyrotropin releasing factor (TRH-R), Vasopressin V1a, V1b and V2,Visual pigments (opsins and rhodopsin), Proto-oncogene mas,Caenorhabditis elegans putative receptors C06G4.5, C38C10.1, C43C3.2,T27D1.3 and ZC84.4, Three putative receptors encoded in the genome ofcytomegalovirus: US27, US28, and UL33., ECRF3, a putative receptorencoded in the genome of herpesvirus saimiri.

[0220] The structure of all GPCRs are thought to be identical. They haveseven hydrophobic regions, each of which most probably spans themembrane. The N-terminus is located on the extracellular side of themembrane and is often glycosylated, while the C-terminus is cytoplasmicand generally phosphorylated. Three extracellular loops alternate withthree intracellular loops to link the seven transmembrane regions. Most,but not all of these receptors, lack a signal peptide. The mostconserved parts of these proteins are the transmembrane regions and thefirst two cytoplasmic loops. A conserved acidic-Arg-aromatic triplet ispresent in the N-terminal extremity of the second cytoplasmic loop andcould be implicated in the interaction with G proteins.

[0221] The putative consensus sequence for GPCRs comprises the conservedtriplet and also spans the major part of the third transmembrane helix,and is as follows:[GSTALIVMFYWC]-[GSTANCPDE]-{EDPKRH}-x(2)-[LIVMNQGA]-x(2)-[LIVMFT]-[GSTANC]-[LIVMFYWSTAC]-[DENH]-R-[FYWCSH]-x(2)-[LIVM],where “X” represents any amino acid.

[0222] Additional information relating to G-protein coupled receptorsmay be found in reference to the following publications: Strosberg A.D., Eur. J. Biochem. 196:1-10(1991); Kerlavage A. R., Curr. Opin.Struct. Biol. 1:394-401(1991); Probst W. C., Snyder L. A., Schuster D.I., Brosius J., Sealfon S. C., DNA Cell Biol. 11:1-20(1992); Savarese T.M., Fraser C. M., Biochem. J. 283:1-9(1992); Branchek T., Curr. Biol.3:315-317(1993); Stiles G. L., J. Biol. Chem. 267:6451-6454(1992);Friell T., Kobilka B. K., Lefkowitz R. J., Caron M. G., Trends Neurosci.11:321-324(1988); Stevens C. F., Curr. Biol. 1:20-22(1991); Sakurai T.,Yanagisawa M., Masaki T., Trends Pharmacol. Sci. 13:103-107(1992);Salesse R., Remy J. J., Levin J. M., Jallal B., Garnier J., Biochimie73:109-120(1991); Lancet D., Ben-Arie N., Curr. Biol. 3:668-674(1993);Uhl G. R., Childers S., Pasternak G., Trends Neurosci. 17:89-93(1994);Barnard E. A., Burnstock G., Webb T. E., Trends Pharmacol. Sci.15:67-70(1994); Applebury M. L., Hargrave P. A., Vision Res.26:1881-1895(1986); Attwood T. K., Eliopoulos E. E., Findlay J. B. C.,Gene 98:153-159(1991); http://www.gcrdb.uthscsa.edu/; andhttp://swift.embl-heidelberg.de/7m/.

[0223] In preferred embodiments, the following G-protein coupledreceptors signature polypeptide is encompassed by the present invention:TCAYLAGLSMLSTVSTERCLSVLWPIW (SEQ ID NO:104). Polynucleotides encodingthis polypeptide are also provided. The present invention alsoencompasses the use of the Gene 7 G-protein coupled receptors signaturepolypeptide as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0224] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:7 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 1983 ofSEQ ID NO:7, b is an integer between 15 to 1997, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:7,and where b is greater than or equal to a+14.

[0225] In one embodiment, a Gene 7 polypeptide comprises a portion ofthe amino sequence depicted in FIGS. 13A-C and/or FIG. 14. In anotherembodiment, a Gene 7 polypeptide comprises at least 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the aminosequence depicted in FIGS. 13A-C and/or FIG. 14. In further embodiments,the following Gene 7 polypeptide fragments are specifically excludedfrom the present invention: VSTERCLSVLWPIWYRC (SEQ ID NO:169);HLSAVVCVLLWALSLL (SEQ ID NO:170);ADFLFLCFQIINCLVYLSNFFCSISINFPSFFTTVMTCAYLAGLSMLSTVSTERCLSVLWPIWYRCRRPRHLSAVVCVLLWALSLLLSILEGKFCGFLFSDGD (SEQ ID NO:171);TILLTVLVFLLCGLPFGIQ (SEQ ID NO:172); LNSSANPIIYFFVGSFR (SEQ ID NO:173);MDSTIPVLGTELTPINGREETPCYKQTLSFTGLTCIVSLVALTGNAVVLWLLGCRMRRNAVSIYILNLVAADFLFL (SEQ ID NO:174);FDFITAAWLIFLFMVLCGSSLALLVRILCGSRGLPLTRLYLTILLTVLV (SEQ ID NO:175);LLCGLPFGIQWFLILWIWKDSDVLFCHIHPVSVVLSSLNSSANPIIYFFVGSFRK QWR (SEQ IDNO:176); PILKLALQRALQDIAEVD (SEQ ID NO:177); LTPINGREETPCYKQTLSFT (SEQID NO:178); and/or LTGNAVVLWLLGCRMRRNAVSIYILNL (SEQ ID NO:179).

[0226] Features of the Polypeptide Encoded by Gene No:10

[0227] The determined nucleotide sequence of the Gene 10 cDNA in FIGS.19A-B (SEQ ID NO:10) contains an open reading frame encoding a proteinof about 311 amino acid residues, with a deduced molecular weight ofabout 34.9 kDa. The amino acid sequence of the predicted Gene 10polypeptide is shown in FIGS. 19A-B and in FIG. 20 (SEQ ID NO:23).

[0228] The Gene 10 polypeptide was predicted to comprise seventransmembrane domains (TM1 to TM7) located from about amino acid 25 toabout amino acid 50 (TM1; SEQ ID NO:105); from about amino acid 61 toabout amino acid 83 (TM2;

[0229] SEQ ID NO:106); from about amino acid 97 to about amino acid 120(TM3; SEQ ID NO: 107); from about amino acid 140 to about amino acid 158(TM4; SEQ ID NO: 108); from about amino acid 197 to about amino acid 226(TM5; SEQ ID NO: 109); from about amino acid 244 to about amino acid 265(TM6; SEQ ID NO:110); and/or from about amino acid 273 to about aminoacid 292 (TM7; SEQ ID NO:11I) of SEQ ID NO:23 (FIGS. 19A-B and FIG. 20).In this context, the term “about” may be construed to mean 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminusof the above referenced transmembrane domain polypeptides.

[0230] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention:LPLFLLFLGIYVVTVVGNLGMTTLIW (SEQ ID NO:105), YFLSSLSFIDFCHSTVITPKMLV (SEQID NO:106), CMTQLYFFLVFAIAECHMLAAMAY (SEQ ID NO:107),ACFSLILGVYIIGLVCASV (SEQ ID NO:108), LLILCVGAFNILVPSLTILCSYIFIIASIL (SEQID NO:109), HMLAVVIFFGSAAFMYLQPSSI (SEQ ID NO:110), and/orVSSVFYTIIVPMLNPLIYSL (SEQ ID NO:111). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these Gene 10 transmembrane domain polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0231] In preferred embodiments, the present invention encompasses theuse of N-terminal deletions, C-terminal deletions, or any combination ofN-terminal and C-terminal deletions of any one or more of the Gene 10TM1 thru TM7 transmembrane domain polypeptides as antigenic and/orimmunogenic epitopes.

[0232] In preferred embodiments, the present invention also encompassesthe use of N-terminal deletions, C-terminal deletions, or anycombination of N-terminal and C-terminal deletions of any one or more ofthe amino acids intervening (i.e., GPCR extracellular or intracellularloops) any pair of Gene 10 TM1 thru TM7 transmembrane domainpolypeptides, and/or the amino acids intervening any pair of the Gene 10TM1 thru TM7 transmembrane domain polypeptides themselves, as antigenicand/or immunogenic epitopes.

[0233] The coding region of the Gene 10 polynucleotide is predicted tobe from nucleotide 18 to nucleotide 950 of SEQ ID NO:10 as shown inFIGS. 19A-B, and the polypeptide corresponding to amino acids 1 thru 311of SEQ ID NO:23. The present invention encompasses the polynucleotideencompassing the entire coding region of Gene 10.

[0234] Alternatively, the present invention encompasses a polynucleotidelacking the initiating start codon, in addition to, the resultingencoded polypeptide of Gene 10. Specifically, the present inventionencompasses the polynucleotide corresponding to nucleotides 21 thru 950of SEQ ID NO:10, and the polypeptide corresponding to amino acids 2 thru311 of SEQ ID NO:23. Also encompassed are recombinant vectors comprisingsaid encoding sequence, and host cells comprising said vector.

[0235] In preferred embodiments, the following N-terminal Gene 10deletion polypeptides are encompassed by the present invention: M1-L311,S2-L311, G3-L311, E4-L311, N5-L311, N6-L311, S7-L311, S8-L311, V9-L311,T1O-L311, E11-L311, F12-L311, 113-L311, L14-L311, A15-L311, G16-L311,L17-L311, S18-L311, E19-L311, Q20-L311, P21-L311, E22-L311, L23-L311,Q24-L311, L25-L311, P26-L311, L27-L311, F28-L311, L29-L311, L30-L311,F3i-L311, L32-L311, G33-L311, I34-L311, Y35-L311, V36-L311, V37-L311,T38-L311, V39-L311, V40-L311, G41-L311, N42-L311, L43-L311, G44-L311,M45-L311, T46-L311, T47-L311, L48-L311, I49-L311, W50-L311, L51-L311,S52-L311, S53-L311, H54-L311, L55-L311, H56-L311, T57-L311, P58-L311,M59-L311, Y60-L311, Y61-L311, F62-L311, L63-L311, S64-L311, S65-L311,L66-L311, S67-L311, F68-L311, 169-L311, D70-L311, F71-L311, C72-L311,H73-L311, S74-L311, T75-L311, V76-L311, 177-L311, T78-L311, P79-L311,K80-L311, M81-L311, L82-L311, V83-L311, N84-L311, F85-L311, V86-L311,T87-L311, E88-L311, K89-L311, N90-L311, 191-L311, 192-L311, S93-L311,Y94-L311, P95-L311, E96-L311, C97-L311, M98-L311, T99-L311, Q100-L311,L101-L311, Y102-L311, F103-L311, F104-L311, L105-L311, V106-L311,F107-L311, A108-L311, I109-L311, A110-L311, E111-L311, C112-L311,H113-L311, M114-L311, L115-L311, A116-L311, A117-L311, M118-L311,A119-L311, Y120-L311, D121-L311, R122-L311, Y123-L311, M124-L311,A125-L311, I126-L311, C127-L311, S128-L311, P129-L311, L130-L311,L131-L311, Y132-L311, S133-L311, V134-L311, I135-L311, I136-L311,S137-L311, N138-L311, K139-L311, A140-L311, C141-L311, F142-L311,S143-L311, L144-L311, I145-L311, L146-L311, G147-L311, V148-L311,Y149-L311, I150-L311, I151-L311, G152-L311, L153-L311, V154-L311,C155-L311, A156-L311, S157-L311, V158-L311, H159-L311, T160-L311,G161-L311, C162-L311, M163-L311, F164-L311, R165-L311, V166-L311,Q167-L311, F168-L311, C169-L311, K170-L311, F171-L311, D172-L311,L173-L311, I174-L311, N175-L311, H176-L311, Y177-L311, F178-L311,C179-L311, D180-L311, L181-L311, L182-L311, P183-L311, L184-L311,L185-L311, K186-L311, L187-L311, S188-L311, C189-L311, S190-L311,S191-L311, I192-L311, Y193-L311, V194-L311, N195-L311, K196-L311,L197-L311, L198-L311, I199-L311, L200-L311, C201-L311, V202-L311,G203-L311, A204-L311, F205-L311, N206-L311, I207-L311, L208-L311,V209-L311, P210-L311, S211-L311, L212-L311, T213-L311, I214-L311,L215-L311, C216-L311, S217-L311, Y218-L311, I219-L311, F220-L311,I221-L311, I222-L311, A223-L311, S224-L311, I225-L311, L226-L311,H227-L311, I228-L311, R229-L311, S230-L311, T231-L311, E232-L311,G233-L311, R234-L311, S235-L311, K236-L311, A237-L311, F238-L311,S239-L311, T240-L311, C241-L311, S242-L311, S243-L311, H244-L311,M245-L311, L246-L311, A247-L311, V248-L311, V249-L311, I250-L311,F251-L311, F252-L311, G253-L311, S254-L311, A255-L311, A256-L311,F257-L311, M258-L311, Y259-L311, L260-L311, Q261-L311, P262-L311,S263-L311, S264-L311, I265-L311, S266-L311, S267-L311, M268-L311,D269-L311, Q270-L311, G271-L311, K272-L311, V273-L311, S274-L311,S275-L311, V276-L311, F277-L311, Y278-L311, T279-L311, I280-L311,I281-L311, V282-L311, P283-L311, M284-L311, L285-L311, N286-L311,P287-L311, L288-L311, I289-L311, Y290-L311, S291-L311, L292-L311,R293-L311, N294-L311, K295-L311, D296-L311, V297-L311, H298-L311,V299-L311, S300-L311, L301-L311, K302-L311, K303-L311, M304-L311, and/orL305-L311 of SEQ ID NO:23. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these N-terminal Gene 10 deletion polypeptides as immunogenicand/or antigenic epitopes as described elsewhere herein.

[0236] In preferred embodiments, the following C-terminal Gene 10deletion polypeptides are encompassed by the present invention: M1-L311,M1-L310, M1-T309, M1-R308, M1-R307, M1-Q306, M1-L305, M1-M304, M1-K303,M1-K302, M1-L301, M1-S300, M1-V299, M1-H298, M1-V297, M1-D296, M1-K295,M1-N294, M1-R293, M1-L292, M1-S291, M1-Y290, M1-I289, M1-L288, M1-P287,M1-N286, M1-L285, M1-M284, M1-P283, M1-V282, M1-I281, M1-I280, M1-T279,M1-Y278, M1-F277, M1-V276, M1-S275, M1-S274, M1-V273, M1-K272, M1-G271,M1-Q270, M1-D269, M1-M268, M1-S267, M1-S266, M1-1265, M1-S264, M1-S263,M1-P262, M1-Q261, M1-L260, M1-Y259, M1-M258, M1-F257, M1-A256, M1-A255,M1-S254, M1-G253, M1-F252, M1-F251, M1-I250, M1-V249, M1-V248, M1-A247,M1-L246, M1-M245, M1-H244, M1-S243, M1-S242, M1-C241, M1-T240, M1-S239,M1-F238, M1-A237, M1-K236, M1-S235, M1-R234, M1-G233, M1-E232, M1-T231,M1-S230, M1-R229, M1-I228, M1-H227, M1-L226, M1-I225, M1-S224, M1-A223,M1-I222, M1-I221, M1-F220, M1-1219, M1-Y218, M1-S217, M1-C216, M1-L215,M1-I214, M1-T213, M1-L212, M1-S211, M1-P210, M1-V209, M1-L208, M1-I207,M1-N206, M1-F205, M1-A204, M1-G203, M1-V202, M1-C201, M1-L200, M1-I199,M1-L198, M1-L197, M1-K196, M1-N195, M1-V194, M1-Y193, M1-I192, M1-S191,M1-S190, M1-C189, M1-S188, M1-L187, M1-K186, M1-L185, M1-L184, M1-P183,M1-L182, M1-L181, M1-D180, M1-C179, M1-F178, M1-Y177, M1-H176, M1-N175,M1-1174, M1-L173, M1-D172, M1-F171, M1-K170, M1-C169, M1-F168, M1-Q167,M1-V166, M1-R165, M1-F164, M1-M163, M1-C162, M1-G161, M1-T160, M1-H159,M1-V158, M1-S157, M1-A156, M1-C155, M1-V154, M1-L153, M1-G152, M1-I151,M1-I150, M1-Y149, M1-V148, M1-G147, M1-L146, M1-1145, M1-L144, M1-S143,M1-F142, M1-C141, M1-A140, M1-K139, M1-N138, M1-S137, M1-I136, M1-I135,M1-V134, M1-S133, M1-Y132, M1-L131, M1-L130, M1-P129, M1-S128, M1-C127,M1-I126, M1-A125, M1-M124, M1-Y123, M1-R122, M1-D121, M1-Y120, M1-A119,M1-M118, M1-A117, M1-A116, M1-L115, M1-M114, M1-H113, M1-C112, M1-E111,M1-A110, M1-I109, M1-A108, M1-F107, M1-V106, M1-L105, M1-F104, M1-F103,M1-Y102, M1-L101, M1-Q100, M1-T99, M1-M98, M1-C97, M1-E96, M1-P95,M1-Y94, M1-S93, M1-192, M1-191, M1-N90, M1-K89, M1-E88, M1-T87, M1-V86,M1-F85, M1-N84, M1-V83, M1-L82, M1-M81, M1-K80, M1-P79, M1-T78, M1-I77,M1-V76, M1-T75, M1-S74, M1-H73, M1-C72, M1-F71, M1-D70, M1-I69, M1-F68,M1-S67, M1-L66, M1-S65, M1-S64, M1-L63, M1-F62, M1-Y61, M1-Y60, M1-M59,M1-P58, M1-T57, M1-H56, M1-L55, M1-H54, M1-S53, M1-S52, M1-L51, M1-W50,M1-I49, M1-L48, M1-T47, M1-T46, M1-M45, M1-G44, M1-L43, M1-N42, M1-G41,M1-V40, M1-V39, M1-T38, M1-V37, M1-V36, M1-Y35, M1-I34, M1-G33, M1-L32,M1-F31, M1-L30, M1-L29, M1-F28, M1-L27, M1-P26, M1-L25, M1-Q24, M1-L23,M1-E22, M1-P21, M1-Q20, M1-E19, M1-S18, M1-L17, M1-G16, M1-A15, M1-L14,M1-I13, M1-F12, M1-E11, M1-T10, M1-V9, M1-S8, and/or M1-S7 of SEQ IDNO:23. Polynucleotide sequences encoding these polypeptides are alsoprovided. The present invention also encompasses the use of theseC-terminal Gene 10 deletion polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0237] The Gene 10 polypeptides of the present invention were determinedto comprise several phosphorylation sites based upon the Motif algorithm(Genetics Computer Group, Inc.). The phosphorylation of such sites mayregulate some biological activity of the Gene 10 polypeptide. Forexample, phosphorylation at specific sites may be involved in regulatingthe proteins ability to associate or bind to other molecules (e.g.,proteins, ligands, substrates, DNA, etc.). In the present case,phosphorylation may modulate the ability of the Gene 10 polypeptide toassociate with other polypeptides, particularly cognate ligand for Gene10, or its ability to modulate certain cellular signal pathways.

[0238] The Gene 10 polypeptide was predicted to comprise five PKCphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem.260:12492-12499(1985); which are hereby incorporated by referenceherein.

[0239] In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: HSTVITPKMLVNF(SEQ ID NO:112), LVNFVTEKNIISY (SEQ ID NO:113), YSVIISNKACFSL (SEQ IDNO:114), NPLIYSLRNKDVH (SEQ ID NO:115), and/or KDVHVSLKKMLQR (SEQ IDNO:116). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of the Gene 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, or 13 PKC phosphorylation site polypeptidesas immunogenic and/or antigenic epitopes as described elsewhere herein.

[0240] The Gene 10 polypeptide was predicted to comprise four caseinkinase II phosphorylation sites using the Motif algorithm (GeneticsComputer Group, Inc.). Casein kinase II (CK-2) is a proteinserine/threonine kinase whose activity is independent of cyclicnucleotides and calcium. CK-2 phosphorylates many different proteins.The substrate specificity [1] of this enzyme can be summarized asfollows: (1) Under comparable conditions Ser is favored over Thr.; (2)An acidic residue (either Asp or Glu) must be present three residuesfrom the C-terminal of the phosphate acceptor site; (3) Additionalacidic residues in positions +1, +2, +4, and +5 increase thephosphorylation rate. Most physiological substrates have at least oneacidic residue in these positions; (4) Asp is preferred to Glu as theprovider of acidic determinants; and (5) A basic residue at theN-terminal of the acceptor site decreases the phosphorylation rate,while an acidic one will increase it.

[0241] A consensus pattern for casein kinase II phosphorylations site isas follows: [ST]-x(2)-[DE], wherein ‘x’ represents any amino acid, and Sor T is the phosphorylation site.

[0242] Additional information specific to casein kinase IIphosphorylation sites may be found in reference to the followingpublication: Pinna L. A., Biochim. Biophys. Acta 1054:267-284(1990);which is hereby incorporated herein in its entirety.

[0243] In preferred embodiments, the following casein kinase IIphosphorylation site polypeptide is encompassed by the presentinvention: GENNSSVTEFILAG (SEQ ID NO:117), FLSSLSFIDFCHST (SEQ IDNO:118), EKNIISYPECMTQL (SEQ ID NO: 119), and/or QPSSISSMDQGKVS (SEQ IDNO:120). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of this casein kinase IIphosphorylation site polypeptide as an immunogenic and/or antigenicepitope as described elsewhere herein.

[0244] The Gene 10 polypeptide has been shown to comprise twoglycosylation sites according to the Motif algorithm (Genetics ComputerGroup, Inc.). As discussed more specifically herein, proteinglycosylation is thought to serve a variety of functions including:augmentation of protein folding, inhibition of protein aggregation,regulation of intracellular trafficking to organelles, increasingresistance to proteolysis, modulation of protein antigenicity, andmediation of intercellular adhesion.

[0245] Asparagine glycosylation sites have the following consensuspattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site.However, it is well known that that potential N-glycosylation sites arespecific to the consensus sequence Asn-Xaa-Ser/Thr. However, thepresence of the consensus tripeptide is not sufficient to conclude thatan asparagine residue is glycosylated, due to the fact that the foldingof the protein plays an important role in the regulation ofN-glycosylation. It has been shown that the presence of proline betweenAsn and Ser/Thr will inhibit N-glycosylation; this has been confirmed bya recent statistical analysis of glycosylation sites, which also showsthat about 50% of the sites that have a proline C-terminal to Ser/Thrare not glycosylated. Additional information relating to asparagineglycosylation may be found in reference to the following publications,which are hereby incorporated by reference herein: Marshall R. D., Annu.Rev. Biochem. 41:673-702(1972); Pless D. D., Lennarz W. J., Proc. Natl.Acad. Sci. U.S.A. 74:134-138(1977); Bause E., Biochem. J.209:331-336(1983); Gavel Y., von Heijne G., Protein Eng.3:433-442(1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem.265:11397-11404(1990).

[0246] In preferred embodiments, the following asparagine glycosylationsite polypeptides are encompassed by the present invention:MSGENNSSVTEFI (SEQ ID NO: 121), and/or MSGENNSSVTEFIL (SEQ ID NO:122).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these Gene 10 asparagineglycosylation site polypeptide as immunogenic and/or antigenic epitopesas described elsewhere herein.

[0247] The Gene 10 polypeptide was predicted to comprise oneN-myristoylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). An appreciable number of eukaryotic proteins are acylatedby the covalent addition of myristate (a C14-saturated fatty acid) totheir N-terminal residue via an amide linkage. The sequence specificityof the enzyme responsible for this modification, myristoyl CoA:proteinN-myristoyl transferase (NMT), has been derived from the sequence ofknown N-myristoylated proteins and from studies using syntheticpeptides. The specificity seems to be the following: i.) The N-terminalresidue must be glycine; ii.) In position 2, uncharged residues areallowed; iii.) Charged residues, proline and large hydrophobic residuesare not allowed; iv.) In positions 3 and 4, most, if not all, residuesare allowed; v.) In position 5, small uncharged residues are allowed(Ala, Ser, Thr, Cys, Asn and Gly). Serine is favored; and vi.) Inposition 6, proline is not allowed.

[0248] A consensus pattern for N-myristoylation is as follows:G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}, wherein ‘x’ represents any amino acid,and G is the N-myristoylation site.

[0249] Additional information specific to N-myristoylation sites may befound in reference to the following publication: Towler D. A., Gordon J.I., Adams S. P., Glaser L., Annu. Rev. Biochem. 57:69-99(1988); andGrand R. J. A., Biochem. J. 258:625-638(1989); which is herebyincorporated herein in its entirety.

[0250] In preferred embodiments, the following N-myristoylation sitepolypeptides are encompassed by the present invention: GVYIIGLVCASVHTGC(SEQ ID NO: 123). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of theseN-myristoylation site polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0251] Moreover, in confirmation of Gene 10 representing a novel GPCR,the Gene 10 polypeptide was predicted to comprise a G-protein coupledreceptor motif using the Motif algorithm (Genetics Computer Group,Inc.). G-protein coupled receptors (also called R7G) are an extensivegroup of hormones, neurotransmitters, odorants and light receptors whichtransduce extracellular signals by interaction with guaninenucleotide-binding (G) proteins. Some examples of receptors that belongto this family are provided as follows: 5-hydroxytryptamine (serotonin)1A to 1F, 2A to 2C, 4, 5A, 5B, 6 and 7, Acetylcholine, muscarinic-type,M1 to M5, Adenosine A1, A2A, A2B and A3, Adrenergic alpha-1A to —IC;alpha-2A to −2D; beta-1 to -3, Angiotensin II types I and II, Bombesinsubtypes 3 and 4, Bradykinin B 1 and B2, c3a and C5a anaphylatoxin,Cannabinoid CB1 and CB2, Chemokines C-C CC-CKR-1 to CC-CKR-8, ChemokinesC-X-C CXC-CKR-1 to CXC-CKR-4, Cholecystokinin-A andcholecystokinin-B/gastrin, Dopamine D1 to D5, Endothelin ET-a and ET-b,fMet-Leu-Phe (fMLP) (N-formyl peptide), Follicle stimulating hormone(FSH-R), Galanin, Gastrin-releasing peptide (GRP-R),Gonadotropin-releasing hormone (GNRH-R), Histamine H1 and H2 (gastricreceptor I), Lutropin-choriogonadotropic hormone (LSH-R), MelanocortinMC1R to MC5R, Melatonin, Neuromedin B (NMB-R), Neuromedin K (NK-3R),Neuropeptide Y types 1 to 6, Neurotensin (NT-R), Octopamine (tyramine)from insects, Odorants, Opioids delta-, kappa- and mu-types, Oxytocin(OT-R), Platelet activating factor (PAF-R), Prostacyclin, ProstaglandinD2, Prostaglandin E2, EP1 to EP4 subtypes, Prostaglandin F2,Purinoreceptors (ATP), Somatostatin types 1 to 5, Substance-K (NK-2R),Substance-P (NK-1R), Thrombin, Thromboxane A2, Thyrotropin (TSH-R),Thyrotropin releasing factor (TRH-R), Vasopressin V1a, V1b and V2,Visual pigments (opsins and rhodopsin), Proto-oncogene mas,Caenorhabditis elegans putative receptors C06G4.5, C38C10. 1, C43C3.2,T27D1.3 and ZC84.4, Three putative receptors encoded in the genome ofcytomegalovirus: US27, US28, and UL33., ECRF3, a putative receptorencoded in the genome of herpesvirus saimiri.

[0252] The structure of all GPCRs are thought to be identical. They haveseven hydrophobic regions, each of which most probably spans themembrane. The N-terminus is located on the extracellular side of themembrane and is often glycosylated, while the C-terminus is cytoplasmicand generally phosphorylated. Three extracellular loops alternate withthree intracellular loops to link the seven transmembrane regions. Most,but not all of these receptors, lack a signal peptide. The mostconserved parts of these proteins are the transmembrane regions and thefirst two cytoplasmic loops. A conserved acidic-Arg-aromatic triplet ispresent in the N-terminal extremity of the second cytoplasmic loop andcould be implicated in the interaction with G proteins.

[0253] The putative consensus sequence for GPCRs comprises the conservedtriplet and also spans the major part of the third transmembrane helix,and is as follows:[GSTALIVMFYWC]-[GSTANCPDE]-{EDPKRH}-x(2)-[LIVMNQGA]-x(2)-[LIVMFT]-[GSTANC]-[LIVMFYWSTAC]-[DENH]-R-[FYWCSH]-x(2)-[LIVM],where “X” represents any amino acid.

[0254] Additional information relating to G-protein coupled receptorsmay be found in reference to the following publications: Strosberg A.D., Eur. J. Biochem. 196:1-10(1991); Kerlavage A. R., Curr. Opin.Struct. Biol. 1:394-401(1991); Probst W. C., Snyder L. A., Schuster D.I., Brosius J., Sealfon S. C., DNA Cell Biol. 11:1-20(1992); Savarese T.M., Fraser C. M., Biochem. J. 283:1-9(1992); Branchek T., Curr. Biol.3:315-317(1993); Stiles G. L., J. Biol. Chem. 267:6451-6454(1992);Friell T., Kobilka B. K., Lefkowitz R. J., Caron M. G., Trends Neurosci.11:321-324(1988); Stevens C. F., Curr. Biol. 1:20-22(1991); Sakurai T.,Yanagisawa M., Masaki T., Trends Pharmacol. Sci. 13:103-107(1992);Salesse R., Remy J. J., Levin J. M., Jallal B., Garnier J., Biochimie73:109-120(1991); Lancet D., Ben-Arie N., Curr. Biol. 3:668-674(1993);Uhl G. R., Childers S., Pasternak G., Trends Neurosci. 17:89-93(1994);Barnard E. A., Burnstock G., Webb T. E., Trends Pharmacol. Sci.15:67-70(1994); Applebury M. L., Hargrave P. A., Vision Res.26:1881-1895(1986); Attwood T. K., Eliopoulos E. E., Findlay J. B. C.,Gene 98:153-159(1991); http://www.gcrdb.uthscsa.edu/; andhttp://swift.embl-heidelberg.de/7tm/.

[0255] In preferred embodiments, the following G-protein coupledreceptors signature polypeptide is encompassed by the present invention:LVFAIAECHMLAAMAYDRYMAICSPLL (SEQ ID NO:123). Polynucleotides encodingthis polypeptide are also provided. The present invention alsoencompasses the use of the Gene 10 G-protein coupled receptors signaturepolypeptide as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0256] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:10 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 947 ofSEQ ID NO:10, b is an integer between 15 to 961, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ IDNO:10, and where b is greater than or equal to a+14.

[0257] In one embodiment, a Gene 10 polypeptide comprises a portion ofthe amino sequence depicted in FIGS. 19A-B and/or FIG. 20. In anotherembodiment, a Gene 10 polypeptide comprises at least 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the aminosequence depicted in FIGS. 19A-B and/or FIG. 20. In further embodiments,the following Gene 10 polypeptide fragments are specifically excludedfrom the present invention: INHYFCDLLPLL (SEQ ID NO:180); SKAFSTCSSH(SEQ ID NO:181); NPLIYSLRNKDV (SEQ ID NO:182); STEGRSKAFSTCSSH (SEQ IDNO:183); FFGSAAFMYLQPSS (SEQ ID NO:184); IVPMLNPLIYSLRNKDV (SEQ IDNO:185); SSMDQGKVSSVFY (SEQ ID NO:186); and/or VPMLNPLIYSLRNKDV (SEQ IDNO:187).

[0258] Features of the Polypeptide Encoded by Gene No:13

[0259] The determined nucleotide sequence of the Gene 13 (also referredto as Gene 13; GPCR-P20; and GPCR-P151) cDNA in FIGS. 25A-B (SEQ IDNO:13) contains an open reading frame encoding a protein of about 295amino acid residues, with a deduced molecular weight of about 32.1 kDa.The amino acid sequence of the predicted Gene 13 polypeptide is shown inFIGS. 25A-B and in FIG. 26 (SEQ ID NO:26).

[0260] The Gene 13 polypeptide was determined to share 29.7% amino acidsequence identity and 41.6% amino acid sequence similarity with thehuman GPCR protein human_hypothetical 1 (SEQ ID NO:149); and to share29.7% amino acid sequence identity and 41.6% amino acid sequencesimilarity with the human GPCR protein human_hypothetical 2 (SEQ IDNO:150) as shown in FIGS. 40A-40E.

[0261] The Gene 13 polypeptide was predicted to comprise seventransmembrane domains (TM1 to TM7) located from about amino acid 19 toabout amino acid 37 (TM1; SEQ ID NO:125); from about amino acid 65 toabout amino acid 84 (TM2; SEQ ID NO:126); from about amino acid 98 toabout amino acid 118 (TM3; SEQ ID NO:127); from about amino acid 139 toabout amino acid 157 (TM4; SEQ ID NO:128); from about amino acid 182 toabout amino acid 199 (TM5; SEQ ID NO:129); from about amino acid 227 toabout amino acid 251 (TM6; SEQ ID NO:130); and/or from about amino 265to about amino acid 283 (TM7; SEQ ID NO: 131) of SEQ ID NO:26 (FIGS.25A-B and FIG. 26). In this context, the term “about” may be construedto mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond theN-Terminus and/or C-terminus of the above referenced transmembranedomain polypeptides.

[0262] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention:LLIIIFFFYLLELVHAASL (SEQ ID NO: 125), FVSGTEPEDGYSTVTLNVIR (SEQ IDNO:126), IDSDPDGDLAFTSGNITFEIG (SEQ ID NO:127), AFSVSVLSVSSGSLGAHIN (SEQID NO:128), KVEEATQNITLSIIRLKG (SEQ ID NO:129),ATQGRDYIPASGFALFGANQSEATI (SEQ ID NO:130), and/or ESVFIELLNSTLVAKVQSR(SEQ ID NO:131). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of these Gene13 transmembrane domain polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0263] In preferred embodiments, the present invention encompasses theuse of N-terminal deletions, C-terminal deletions, or any combination ofN-terminal and C-terminal deletions of any one or more of the Gene 13TM1 thru TM7 transmembrane domain polypeptides as antigenic and/orimmunogenic epitopes.

[0264] In preferred embodiments, the present invention also encompassesthe use of N-terminal deletions, C-terminal deletions, or anycombination of N-terminal and C-terminal deletions of any one or more ofthe amino acids intervening (i.e., GPCR extracellular or intracellularloops) any pair of Gene 13 TM1 thru TM7 transmembrane domainpolypeptides, and/or the amino acids intervening any pair of the Gene 13TM1 thru TM7 transmembrane domain polypeptides themselves, as antigenicand/or immunogenic epitopes.

[0265] The coding region of the Gene 13 polynucleotide is predicted tobe from nucleotide 23 to nucleotide 906 of SEQ ID NO:13 as shown inFIGS. 25A-B, and the polypeptide corresponding to amino acids 1 thru 295of SEQ ID NO:26. The present invention encompasses the polynucleotideencompassing the entire coding region of Gene 13.

[0266] Alternatively, the present invention encompasses a polynucleotidelacking the initiating start codon, in addition to, the resultingencoded polypeptide of Gene 13. Specifically, the present inventionencompasses the polynucleotide corresponding to nucleotides 26 thru 906of SEQ ID NO:13, and the polypeptide corresponding to amino acids 2 thru295 of SEQ ID NO:26. Also encompassed are recombinant vectors comprisingsaid encoding sequence, and host cells comprising said vector.

[0267] Expression profiling of Gene 13 indicates it is highly expressedin brain and pituitary (FIG. 42). In particular, Gene 13 was expressedin the following brain sub-regions: amygdala, caudate nucleus, corpuscallosum, cerebellum, hippocampus, thalamus and subtantia nigra (FIG.43). Gene 13 has also been found to be expressed in the heart, kidney,lung, pancreas, prostate, small intestine, spinal cord, testis andthymus.

[0268] Expanded analysis of Gene 13 expression levels by TaqMan™quantitative PCR (see FIG. 44) confirmed that the Gene 13 polypeptide isexpressed in brain, and pituitary (FIG. 42). Gene 13 mRNA was expressedpredominately in the in the nervous system, with lesser amounts found inthe respiratory and endocrine systems. Specifically, Gene 13 wasexpressed at the highest steady state levels in the nucleus accumbens,followed by the pineal and pituitary gland. Expression of Gene 13 wasalso significantly expressed at near equal levels across the cortex,hippocampus, amygdala, and choroid plexus. Expression of Gene 13 wasalso significantly expressed to a lesser extent in in the caudate, thecerebellum and the hypothalamus.

[0269] Collectively the expression data suggests a role for HGRPBMY37 inneural processes that connect, either directly or indirectly, thenucleus accumbens and its ‘reward center’ functions which include, forexample, the release of neurotransmitters such as dopamine, opioidpeptides, serotonin, GABA and the pineal gland, best known for itsinvolvement in the establishment and maintenance of circadian rhythmsand the control of the sleep/wake cycle.

[0270] Polnucleotides and polypeptide of Gene 13, in addition tomodulators thereof, are useful for detecting, treating, and/orameliorating neural disorders, particularly those disorders that affectthe nucleus accumbens, disorders that affect the brains ‘reward center’function, neurotransmitter release disorders, disorders affecting therelease of dopamine, disorders affecting the release of opioid peptides,disorders affecting the release of serotonin, disorders affecting therelease of GABA, pineal gland disorders, disorders affecting theestablishment of circadian rhythms, disorders affecting the maintenanceof circadian rhythms, disorders affecting the control of the sleep/wakecycle.

[0271] The pituitary gland expression may suggest a link between Gene 13and melatonin and pituitary hormone secretions, particularly thoseinvolved in oxytocin secretion during the neuroendocrine response tostressful stimuli. Gene 13 may also have a role in how the melatoninrhythm may affect other neuroendocrine functions such as the nocturnalpattern of hormone secretion, prolactin and cortisol release.

[0272] Polnucleotides and polypeptide of Gene 13, in addition tomodulators thereof, are useful for detecting, treating, and/orameliorating melatonin secretion disorders, pituitary hormone secretiondisorders, oxytocin secretion disorders, disorders affectingneuroendocrine response to stressful stimuli, disorders affectingoxytocin secretion during neuroendocrine response to stressful stimuli,disorders affecting nocturnal patterns of hormone secretion, disordersaffecting the nocturnal hormone secretion of prolactin, disordersaffecting the nocturnal hormone secretion of cortisol, and/or disordersaffecting the nocturnal hormone secretion of growth hormone.

[0273] Gene 13 expression throughout the cortex suggests involvement inthe execution of functions concerned with the organization of behavior,memory and cognitive reasoning. These data suggest modulators of Gene 13function may have utility in a variety of neuro-pathologies, includingresponses to stress, and propensity to develop addictive behaviors, aswell as a vast number of neuroendocrine abnormalities including sleepdisorders.

[0274] The Gene 13 polynucleotide and polypeptide, may be involved in avariety of diseases, disorders and conditions associated with Gene 13activity, which include, but are not limited to, immune-relateddisorders, acute heart failure, hypotension, hypertension, endocrinaldiseases, growth disorders, neuropathic pain, obesity, anorexia, HIVinfections, cancers, bulimia, asthma, Parkinson's disease, osteoporosis,angina pectoris, myocardial infarction, psychotic, metabolic,cardiovascular and neurological disorders. More specifically, thepresent invention is concerned with modulation of the Gene 13polynucleotide and encoded products, particularly in providingtreatments and therapies for relevant diseases. Antagonizing orinhibiting the action of the Gene 13 polynucleotide and polypeptide isespecially encompassed by the present invention.

[0275] The invention further relates to fragments and portions of thenovel Gene 13 nucleic acid sequence and its encoded amino acid sequence(peptides and polypeptides). Preferably, the fragments and portions ofthe Gene 13 polypeptide are functional or active. The invention alsoprovides methods of using the novel Gene 13 polynucleotide sequence andthe encoded Gene 13 polypeptide for diagnosis, genetic screening andtreatment of diseases, disorders, conditions, or syndromes associatedwith Gene 13 and Gene 13 activity and function. The Gene 13polynucleotide and polypeptide, may be involved in a variety ofdiseases, disorders and conditions associated with Gene 13 activity,which include, but are not limited to, immune-related disorders, acuteheart failure, hypotension, hypertension, endocrinal diseases, growthdisorders, neuropathic pain, obesity, anorexia, HIV infections, cancers,bulimia, asthma, Parkinson's disease, osteoporosis, angina pectoris,myocardial infarction, psychotic, metabolic, cardiovascular andneurological disorders. Neurological or nervous system-relatedconditions are particularly relevant.

[0276] The Gene 13 polynucleotide and/or polypeptide of this inventionare useful for diagnosing diseases related to over- or under-expressionof the Gene 13 protein. For example, such Gene 13-associated diseasescan be assessed by identifying mutations in the Gene 13 gene using Gene13 probes or primers, or by determining Gene 13 protein or mRNAexpression levels. A Gene 13 polypeptide is also useful for screeningcompounds which affect activity of the protein. The invention furtherencompasses the polynucleotide encoding the Gene 13 polypeptide and theuse of the Gene 13 polynucleotide or polypeptide, or compositionsthereof, in the screening, diagnosis, treatment, or prevention ofdisorders associated with aberrant or uncontrolled cellular growthand/or function, such as neoplastic diseases (for example, cancers andtumors). Gene 13 probes or primers can be used, for example, to screenfor diseases associated with Gene 13.

[0277] The Gene 13 protein according to this invention may play a rolein cell signaling, in cell cycle regulation, and/or in neurologicaldisorders. The Gene 13 protein may further be involved in neoplastic,cardiovascular, and immunological disorders.

[0278] In one embodiment in accordance with the present invention, thenovel Gene 13 protein may play a role in neoplastic disorders. Anantagonist or inhibitor of the Gene 13 protein may be administered to anindividual to prevent or treat a neoplastic disorder. Such disorders mayinclude, but are not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, and teratocarcinoma, and particularly,cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast,cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. In arelated aspect, an antibody which specifically binds to Gene 13 may beused directly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress the Gene 13 polypeptide.

[0279] In yet another embodiment of the present invention, an antagonistor inhibitory agent of the Gene 13 polypeptide may be administeredtherapeutically to an individual to prevent or treat an immunologicaldisorder. Such disorders may include, but are not limited to, AIDS, HIVinfection, Addison's disease, adult respiratory distress syndrome,allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis,Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjogren's syndrome, and autoimmune thyroiditis;complications of cancer, hemodialysis, extracorporeal circulation;viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections, trauma, and neurological disorders including, but notlimited to, akathesia, Alzheimer's disease, amnesia, amyotrophic lateralsclerosis, bipolar disorder, catatonia, cerebral neoplasms, dementia,depression, Down's syndrome, tardive dyskinesia, dystonias, epilepsy,Huntington's disease, multiple sclerosis, Parkinson's disease, paranoidpsychoses, schizophrenia, and Tourette's disorder.

[0280] A preferred method of treating a Gene 13 associated disease,disorder, syndrome, or condition in a mammal comprises administration ofa modulator, preferably an inhibitor or antagonist, of a Gene 13polypeptide or homologue of the invention, in an amount effective totreat, reduce, and/or ameliorate the symptoms incurred by the Gene13-associated disease, disorder, syndrome, or condition. In someinstances, an agonist or enhancer of a Gene 13 polypeptide or homologueof the invention is administered in an amount effective to treat and/orameliorate the symptoms incurred by a Gene 13-related disease, disorder,syndrome, or condition. In other instances, the administration of anovel Gene 13 polypeptide or homologue thereof pursuant to the presentinvention is envisioned for administration to treat a Gene 13 associateddisease.

[0281] In a preferred embodiment, polynucleotides and polypeptides ofGene 13, in addition to modulators thereof, are useful for treating,preventing, and/or ameliorating the following diseases or disorders,brain development disorders, peripheral nervous system disorders,audiogenic epilepsy, epilepsy, embryonal neurogenesis disorders, eyedevelopment disorders, and neuronal excitability disorders.

[0282] Nervous system diseases, disorders, and/or conditions, which canbe treated, prevented, and/or diagnosed with the compositions of theinvention (e.g., polypeptides, polynucleotides, and/or agonists orantagonists), include, but are not limited to, nervous system injuries,and diseases, disorders, and/or conditions which result in either adisconnection of axons, a diminution or degeneration of neurons, ordemyelination. Nervous system lesions which may be treated, prevented,and/or diagnosed in a patient (including human and non-human mammalianpatients) according to the invention, include but are not limited to,the following lesions of either the central (including spinal cord,brain) or peripheral nervous systems: (1) ischemic lesions, in which alack of oxygen in a portion of the nervous system results in neuronalinjury or death, including cerebral infarction or ischemia, or spinalcord infarction or ischemia; (2) traumatic lesions, including lesionscaused by physical injury or associated with surgery, for example,lesions which sever a portion of the nervous system, or compressioninjuries; (3) malignant lesions, in which a portion of the nervoussystem is destroyed or injured by malignant tissue which is either anervous system associated malignancy or a malignancy derived fromnon-nervous system tissue; (4) infectious lesions, in which a portion ofthe nervous system is destroyed or injured as a result of infection, forexample, by an abscess or associated with infection by humanimmunodeficiency virus, herpes zoster, or herpes simplex virus or withLyme disease, tuberculosis, syphilis; (5) degenerative lesions, in whicha portion of the nervous system is destroyed or injured as a result of adegenerative process including but not limited to degenerationassociated with Parkinson's disease, Alzheimer's disease, Huntington'schorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associatedwith nutritional diseases, disorders, and/or conditions, in which aportion of the nervous system is destroyed or injured by a nutritionaldisorder or disorder of metabolism including but not limited to, vitaminB12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration; (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins; and(9) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myclinolysis.

[0283] In a preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to protect neuralcells from the damaging effects of cerebral hypoxia. According to thisembodiment, the compositions of the invention are used to treat,prevent, and/or diagnose neural cell injury associated with cerebralhypoxia. In one aspect of this embodiment, the polypeptides,polynucleotides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose neural cell injury associated withcerebral ischemia. In another aspect of this embodiment, thepolypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat, prevent, and/or diagnose neural cell injuryassociated with cerebral infarction. In another aspect of thisembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnoseor prevent neural cell injury associated with a stroke. In a furtheraspect of this embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat, prevent,and/or diagnose neural cell injury associated with a heart attack.

[0284] The compositions of the invention which are useful for treatingor preventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture; (2) increased sprouting of neurons in culture or in vivo; (3)increased production of a neuron-associated molecule in culture or invivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, the method set forthin Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increasedsprouting of neurons may be detected by methods known in the art, suchas, for example, the methods set forth in Pestronk et al. (Exp. Neurol.70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 (1981));increased production of neuron-associated molecules may be measured bybioassay, enzymatic assay, antibody binding, Northern blot assay, etc.,using techniques known in the art and depending on the molecule to bemeasured; and motor neuron dysfunction may be measured by assessing thephysical manifestation of motor neuron disorder, e.g., weakness, motorneuron conduction velocity, or functional disability.

[0285] In specific embodiments, motor neuron diseases, disorders, and/orconditions that may be treated, prevented, and/or diagnosed according tothe invention include, but are not limited to, diseases, disorders,and/or conditions such as infarction, infection, exposure to toxin,trauma, surgical damage, degenerative disease or malignancy that mayaffect motor neurons as well as other components of the nervous system,as well as diseases, disorders, and/or conditions that selectivelyaffect neurons such as amyotrophic lateral sclerosis, and including, butnot limited to, progressive spinal muscular atrophy, progressive bulbarpalsy, primary lateral sclerosis, infantile and juvenile muscularatrophy, progressive bulbar paralysis of childhood (Fazio-Londesyndrome), poliomyelitis and the post polio syndrome, and HereditaryMotorsensory Neuropathy (Charcot-Marie-Tooth Disease).

[0286] In preferred embodiments, the following N-terminal Gene 13deletion polypeptides are encompassed by the present invention: M1-N295,E2-N295, G3-N295, L4-N295, F5-N295, S6-N295, K7-N295, S8-N295, C9-N295,S10-N295, L11-N295, A12-N295, F13-N295, S14-N295, L15-N295, I16-N295,C17-N295, K18-N295, L19-N295, L20-N295, I21-N295, I22-N295, I23-N295,F24-N295, F25-N295, F26-N295, Y27-N295, L28-N295, L29-N295, E30-N295,L31-N295, V32-N295, H33-N295, A34-N295, A35-N295, S36-N295, L37-N295,G38-N295, V39-N295, A40-N295, S41-N295, Q42-N295, I43-N295, L44-N295,V45-N295, T46-N295, I47-N295, A48-N295, A49-N295, S50-N295, D51-N295,H52-N295, A53-N295, H54-N295, G55-N295, V56-N295, F57-N295, E58-N295,F59-N295, S60-N295, P61-N295, E62-N295, S63-N295, L64-N295, F65-N295,V66-N295, S67-N295, G68-N295, T69-N295, E70-N295, P71-N295, E72-N295,D73-N295, G74-N295, Y75-N295, S76-N295, T77-N295, V78-N295, T79-N295,L80-N295, N81-N295, V82-N295, I83-N295, R84-N295, H85-N295, H86-N295,G87-N295, T88-N295, L89-N295, S90-N295, P91-N295, V92-N295, T93-N295,L94-N295, H95-N295, W96-N295, N97-N295, I98-N295, D99-N295, S100-N295,D101-N295, P102-N295, D103-N295, G104-N295, D105-N295, L106-N295,A107-N295, F108-N295, T109-N295, S110-N295, G111-N295, N112-N295,I113-N295, T114-N295, F115-N295, E116-N295, I117-N295, G118-N295,Q119-N295, T120-N295, S121-N295, A122-N295, N123-N295, I124-N295,T125-N295, V126-N295, E127-N295L, I128-N295, L129-N295, P130-N295,D131-N295, E132-N295, D133-N295, P134-N295, E135-N295, L136-N295,D137-N295, K138-N295, A139-N295, F140-N295, S141-N295, V142-N295,S143-N295, V144-N295, L145-N295, S146-N295, V147-N295, S148-N295,S149-N295, G150-N295, S151-N295, L152-N295, G153-N295, A154-N295,H155-N295, I156-N295, N157-N295, A158-N295, T159-N295, L160-N295,T161-N295, V162-N295, L163-N295, A164-N295, S165-N295, D166-N295,D167-N295, P168-N295, Y169-N295, G170-N295, I171-N295, F172-N295,I173-N295, F174-N295, S175-N295, E176-N295, K177-N295, N178-N295,R179-N295, P180-N295, V181-N295, K182-N295, V183-N295, E184-N295,E185-N295, A186-N295, T187-N295, Q188-N295, N189-N295, I190-N295,T191-N295, L192-N295, S193-N295, I194-N295, I195-N295, R196-N295,L197-N295, K198-N295, G199-N295, L200-N295, M201-N295, G202-N295,K203-N295, V204-N295, L205-N295, V206-N295, S207-N295, Y208-N295,A209-N295, T210-N295, L211-N295, D212-N295, D213-N295, M214-N295,E215-N295, K216-N295, P217-N295, P218-N295, Y219-N295, F220-N295,P221-N295, P222-N295, N223-N295, L224-N295, A225-N295, R226-N295,A227-N295, T228-N295, Q229-N295, G230-N295, R231-N295, D232-N295,Y233-N295, I234-N295, P235-N295, A236-N295, S237-N295, G238-N295,F239-N295, A240-N295, L241-N295, F242-N295, G243-N295, A244-N295,N245-N295, Q246-N295, S247-N295, E248-N295, A249-N295, T250-N295,I251-N295, A252-N295, I253-N295, S254-N295, I255-N295, L256-N295,D257-N295, D258-N295, D259-N295, E260-N295, P261-N295, E262-N295,R263-N295, S264-N295, E265-N295, S266-N295, V267-N295, F268-N295,I269-N295, E270-N295, L271-N295, L272-N295, N273-N295, S274-N295,T275-N295, L276-N295, V277-N295, A278-N295, K279-N295, V280-N295,Q281-N295, S282-N295, R283-N295, S284-N295, S285-N295, K286-N295,Y287-N295, P288-N295, and/or L289-N295 of SEQ ID NO:26. Polynucleotidesequences encoding these polypeptides are also provided. The presentinvention also encompasses the use of these N-terminal Gene 13 deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0287] In preferred embodiments, the following C-terminal Gene 13deletion polypeptides are encompassed by the present invention: M1-N295,M1-Y294, M1-Y293, M1-Y292, M1-C291, M1-V290, M1-L289, M1-P288, M1-Y287,M1-K286, M1-S285, M1-S284, M1-R283, M1-S282, M1-Q281, M1-V280, M1-K279,M1-A278, M1-V277, M1-L276, M1-T275, M1-S274, M1-N273, M1-L272, M1-L271,M1-E270, M1-I269, M1-F268, M1-V267, M1-S266, M1-E265, M1-S264, M1-R263,M1-E262, M1-P261, M1-E260, M1-D259, M1-D258, M1-D257, M1-L256, M1-I255,M1-S254, M1-I253, M1-A252, M1-I251, M1-T250, M1-A249, M1-E248, M1-S247,M1-Q246, M1-N245, M1-A244, M1-G243, M1-F242, M1-L241, M1-A240, M1-F239,M1-G238, M1-S237, M1-A236, M1-P235, M1-1234, M1-Y233, M1-D232, M1-R231,M1-G230, M1-Q229, M1-T228, M1-A227, M1-R226, M1-A225, M1-L224, M1-N223,M1-P222, M1-P221, M1-F220, M1-Y219, M1-P218, M1-P217, M1-K216, M1-E215,M1-M214, M1-D213, M1-D212, M1-L211, M1-T210, M1-A209, M1-Y208, M1-S207,M1-V206, M1-L205, M1-V204, M1-K203, M1-G202, M1-M201, M1-L200, M1-G199,M1-K198, M1-L197, M1-R196, M1-I195, M1-I194, M1-S193, M1-L192, M1-T191,M1-I190, M1-N189, M1-Q188, M1-T187, M1-A186, M1-E185, M1-E184, M1-V183,M1-K182, M1-V181, M1-P180, M1-R179, M1-N178, M1-K177, M1-E176, M1-S175,M1-F174, M1-I173, M1-F172, M1-I171, M1-G170, M1-Y169, M1-P168, M1-D167,M1-D166, M1-S165, M1-A164, M1-L163, M1-V162, M1-T161, M1-L160, M1-T159,M1-A158, M1-N157, M1-I156, M1-H155, M1-A154, M1-G153, M1-L152, M1-S151,M1-G150, M1-S149, M1-S148, M1-V147, M1-S146, M1-L145, M1-V144, M1-S143,M1-V142, M1-S141, M1-F140, M1-A139, M1-K138, M1-D137, M1-L136, M1-E135,M1-P134, M1-D133, M1-E132, M1-D131, M1-P130, M1-L129, M1-I128, M1-E127,M1-V126, M1-T125, M1I124, M1-N123, M1-A122, M1-S121, M1-T120, M1-Q119,M1-G118, M1-I117, M1-E116, M1-F115, M1-T114, M1-I113, M1-N112, M1-G111,M1-S110, M1-T109, M1-F108, M1-A107, M1-L106, M1-D105, M1-G104, M1-D103,M1-P102, M1-D101, M1-S100, M1-D99, M1-198, M1-N97, M1-W96, M1-H95,M1-L94, M1-T93, M1V92, M1-P91, M1-S90, M1-L89, M1-T88, M1-G87, M1-H86,M1-H85, M1-R84, M1-I83, M1-V82, M1-N81, M1-L80, M1-T79, M1-V78, M1-T77,M1-S76, M1-Y75, M1-G74, M1-D73, M1-E72, M1-P71, M1-E70, M1-T69, M1-G68,M1-S67, M1-V66, M1-F65, M1-L64, M1-S63, M1-E62, M1-P61, M1-S60, M1-F59,M1-E58, M1-F57, M1-V56, M1-G55, M1-H54, M1-A53, M1-H52, M1-D51, M1-S50,M1-A49, M1-A48, M1-I47, M1-T46, M1-V45, M1-L44, M1-I43, M1-Q42, M1-S41,M1-A40, M1-V39, M1-G38, M1-L37, M1-S36, M1-A35, M1-A34, M1-H33, M1-V32,M1-L31, M1-E30, M1-L29, M1-L28, M1-Y27, M1-F26, M1-F25, M1-F24, M1-I23,M1-I22, M1-121, M1-L20, M1-L19, M1-K18, M1-C17, M1-I16, M1-L15, M1-S14,M1-F13, M1-A12, M1-L11, M1-S10, M1-C9, M1-S8, and/or M1-K7 of SEQ IDNO:26. Polynucleotide sequences encoding these polypeptides are alsoprovided. The present invention also encompasses the use of theseC-terminal Gene 13 deletion polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0288] The Gene 13 polypeptides of the present invention were determinedto comprise several phosphorylation sites based upon the Motif algorithm(Genetics Computer Group, Inc.). The phosphorylation of such sites mayregulate some biological activity of the Gene 13 polypeptide. Forexample, phosphorylation at specific sites may be involved in regulatingthe proteins ability to associate or bind to other molecules (e.g.,proteins, ligands, substrates, DNA, etc.). In the present case,phosphorylation may modulate the ability of the Gene 13 polypeptide toassociate with other polypeptides, particularly cognate ligand for Gene13, or its ability to modulate certain cellular signal pathways.

[0289] The Gene 13 polypeptide was predicted to comprise two PKCphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem.260:12492-12499(1985); which are hereby incorporated by referenceherein.

[0290] In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: GIFIFSEKNRPVK(SEQ ID NO:132), and/or KVQSRSSKYPLVC (SEQ ID NO:133). Polynucleotidesencoding these polypeptides are also provided. The present inventionalso encompasses the use of the Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or 13 PKC phosphorylation site polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

[0291] The Gene 13 polypeptide was predicted to comprise five caseinkinase II phosphorylation sites using the Motif algorithm (GeneticsComputer Group, Inc.). Casein kinase II (CK-2) is a proteinserine/threonine kinase whose activity is independent of cyclicnucleotides and calcium. CK-2 phosphorylates many different proteins.The substrate specificity [1] of this enzyme can be summarized asfollows: (1) Under comparable conditions Ser is favored over Thr.; (2)An acidic residue (either Asp or Glu) must be present three residuesfrom the C-terminal of the phosphate acceptor site; (3) Additionalacidic residues in positions +1, +2, +4, and +5 increase thephosphorylation rate. Most physiological substrates have at least oneacidic residue in these positions; (4) Asp is preferred to Glu as theprovider of acidic determinants; and (5) A basic residue at theN-terminal of the acceptor site decreases the phosphorylation rate,while an acidic one will increase it.

[0292] A consensus pattern for casein kinase II phosphorylations site isas follows: [ST]-x(2)-[DE], wherein ‘x’ represents any amino acid, and Sor T is the phosphorylation site.

[0293] Additional information specific to casein kinase IIphosphorylation sites may be found in reference to the followingpublication: Pinna L. A., Biochim. Biophys. Acta 1054:267-284(1990);which is hereby incorporated herein in its entirety.

[0294] In preferred embodiments, the following casein kinase IIphosphorylation site polypeptide is encompassed by the presentinvention: ESLFVSGTEPEDGY (SEQ ID NO:134), LFVSGTEPEDGYST (SEQ IDNO:135), HWNIDSDPDGDLAF (SEQ ID NO:136), LVSYATLDDMEKPP (SEQ ID NO:137),and/or ATIAISILDDDEPE (SEQ ID NO:138). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of this casein kinase II phosphorylation site polypeptide as animmunogenic and/or antigenic epitope as described elsewhere herein.

[0295] The Gene 13 polypeptide has been shown to comprise sixglycosylation sites according to the Motif algorithm (Genetics ComputerGroup, Inc.). As discussed more specifically herein, proteinglycosylation is thought to serve a variety of functions including:augmentation of protein folding, inhibition of protein aggregation,regulation of intracellular trafficking to organelles, increasingresistance to proteolysis, modulation of protein antigenicity, andmediation of intercellular adhesion.

[0296] Asparagine glycosylation sites have the following consensuspattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site.However, it is well known that that potential N-glycosylation sites arespecific to the consensus sequence Asn-Xaa-Ser/Thr. However, thepresence of the consensus tripeptide is not sufficient to conclude thatan asparagine residue is glycosylated, due to the fact that the foldingof the protein plays an important role in the regulation ofN-glycosylation. It has been shown that the presence of proline betweenAsn and Ser/Thr will inhibit N-glycosylation; this has been confirmed bya recent statistical analysis of glycosylation sites, which also showsthat about 50% of the sites that have a proline C-terminal to Ser/Thrare not glycosylated. Additional information relating to asparagineglycosylation may be found in reference to the following publications,which are hereby incorporated by reference herein: Marshall R. D., Annu.Rev. Biochem. 41:673-702(1972); Pless D. D., Lennarz W. J., Proc. Natl.Acad. Sci. U.S.A. 74:134-138(1977); Bause E., Biochem. J.209:331-336(1983); Gavel Y., von Heijne G., Protein Eng.3:433-442(1990); and Miletich J.P., Broze G. J. Jr., J. Biol. Chem.265:11397-11404(1990).

[0297] In preferred embodiments, the following asparagine glycosylationsite polypeptides are encompassed by the present invention:AFTSGNITFEIGQT (SEQ ID NO:139), GQTSANITVEILPD (SEQ ID NO:140),LGAHINATLTVLAS (SEQ ID NO:141), EEATQNITLSIIRL (SEQ ID NO:142),ALFGANQSEATIAI (SEQ ID NO:143), and/or FIELLNSTLVAKVQ (SEQ ID NO:144).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these Gene 13 asparagineglycosylation site polypeptide as immunogenic and/or antigenic epitopesas described elsewhere herein.

[0298] The Gene 13 polypeptide was predicted to comprise fourN-myristoylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). An appreciable number of eukaryotic proteins are acylatedby the covalent addition of myristate (a C14-saturated fatty acid) totheir N-terminal residue via an amide linkage. The sequence specificityof the enzyme responsible for this modification, myristoyl CoA:proteinN-myristoyl transferase (NMT), has been derived from the sequence ofknown N-myristoylated proteins and from studies using syntheticpeptides. The specificity seems to be the following: i.) The N-terminalresidue must be glycine; ii.) In position 2, uncharged residues areallowed; iii.) Charged residues, proline and large hydrophobic residuesare not allowed; iv.) In positions 3 and 4, most, if not all, residuesare allowed; v.) In position 5, small uncharged residues are allowed(Ala, Ser, Thr, Cys, Asn and Gly). Serine is favored; and vi.) Inposition 6, proline is not allowed.

[0299] A consensus pattern for N-myristoylation is as follows:G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}, wherein ‘x’ represents any amino acid,and G is the N-myristoylation site.

[0300] Additional information specific to N-myristoylation sites may befound in reference to the following publication: Towler D. A., Gordon J.I., Adams S. P., Glaser L., Annu. Rev. Biochem. 57:69-99(1988); andGrand R. J. A., Biochem. J. 258:625-638(1989); which is herebyincorporated herein in its entirety.

[0301] In preferred embodiments, the following N-myristoylation sitepolypeptides are encompassed by the present invention: ITFEIGQTSANITVEI(SEQ ID NO:145), LSVSSGSLGAHINATL (SEQ ID NO:146), SSGSLGAHINATLTVL (SEQID NO:147), and/or GFALFGANQSEATIAI (SEQ ID NO:148). Polynucleotidesencoding these polypeptides are also provided. The present inventionalso encompasses the use of these N-myristoylation site polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0302] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:13 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 892 ofSEQ ID NO:13, b is an integer between 15 to 906, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ IDNO:13, and where b is greater than or equal to a+14.

[0303] In one embodiment, a Gene 13 polypeptide comprises a portion ofthe amino sequence depicted in FIGS. 25A-B and/or FIG. 26. In anotherembodiment, a Gene 13 polypeptide comprises at least 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the aminosequence depicted in FIGS. 25A-B and/or FIG. 26. In further embodiments,the following Gene 13 polypeptide fragments are specifically excludedfrom the present invention: ITVEILPDEDPELDKAFSVS (SEQ ID NO:188);LSVSSGSLGAHINATLTV (SEQ ID NO:189);SDDPYGIFIFSEKNRPVKVEEATQNITLSIIRLKGLMGKVLVSYATLDDMEKPPYFPPNLARATQGRDYIPASGFAL (SEQ ID NO:190); GANQSEATIAISI (SEQ ID NO:191);or KGLMGKVLVSYATLDDMEKPPYFPPNLARATQGRDYIPASGFALFGANQSEATIAISILDDDEPERSESVFIELLNSTLVAKVQSRS (SEQ ID NO:192).

[0304] GPCR polynucleotides and/or polypeptides are useful fordiagnosing diseases related to over- or under-expression of GPCRproteins. For example, such GPCR-associated diseases can be assessed byidentifying mutations in a GPCR gene using GPCR probes or primers, or bydetermining GPCR protein or mRNA expression levels. GPCR polypeptidesare also useful for screening compounds which affect activity of theprotein. The invention further encompasses the polynucleotides encodingthe GPCR polypeptides and the use of the GPCR polynucleotides orpolypeptides, or compositions thereof, in the screening, diagnosis,treatment, or prevention of disorders associated with aberrant oruncontrolled cellular growth and/or function, such as neoplasticdiseases (for example, cancers and tumors).

[0305] GPCR probes or primers can be used, for example, to screen fordiseases associated with GPCRs. Table 2 lists the predicted left andright primers, i.e., SEQ ID NOs:27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55 and SEQ ID NOs:28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54 56, respectively, as determined from the disclosedGPCR nucleic acid sequences. Table 3 lists the predicted internalprimers for the GPCR polynucleotides of the present invention (SEQ IDNOs:57-71).

[0306] One embodiment of the present invention encompasses novel GPCRpolypeptides comprising the amino acid sequences of SEQ ID NOs:14-26 asshown in FIGS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26,respectively. More specifically, the sensory GPCR polypeptide of SEQ IDNO:14 is 309 amino acids in length and has 50% amino acid sequenceidentity with the human olfactory receptor 5U1 (SEQ ID NO:72), (FIG.27A). The sensory GPCR polypeptide of SEQ ID NO:15 comprises 331 aminoacids and has 98% sequence identity with human G protein-coupledreceptor 61 (SEQ ID NO:73), (FIG. 28A), and 98% sequence identity with aportion of rabbit G protein-coupled receptor (SEQ ID NO:74), (FIG. 28B).The sensory GPCR polypeptide of SEQ ID NO:16 comprises 317 amino acidsand has 48% sequence identity with olfactory receptor 3′Beta4 of mouse(SEQ ID NO:75), (FIG. 29A). The sensory GPCR polypeptide of SEQ ID NO:17comprises 324 amino acids and has 48% sequence identity with theolfactory receptor 3′Beta1 of mouse (SEQ ID NO:76), (FIG. 30A). Thepolynucleotide encoding the GPCR of SEQ ID NO:17 has been localized tolung on the basis of tissue expression analysis. The sensory GPCRpolypeptide of SEQ ID NO:18 comprises 309 amino acids and has 44%sequence identity with human taste receptor T2R13, family B (SEQ IDNO:77), (FIG. 31). The polynucleotide encoding the GPCR of SEQ ID NO:18has been localized to uterus on the basis of tissue expression analysis.The chemokine GPCR polypeptide of SEQ ID NO:19 comprises 372 amino acidsand has 26% sequence identity with human chemokine receptor 1 (SEQ IDNO:78), (FIG. 32). The human orphan GPCR polypeptide of SEQ ID NO:20comprises 328 amino acids and has 77% sequence identity with human Gprotein coupled receptor hHI7T213 (SEQ ID NO:79), (FIG. 33).The-polynucleotide encoding the GPCR of SEQ ID NO:20 has been found inskull tumor on the basis of tissue expression analysis. The human orphanGPCR polypeptide of SEQ ID NO:21 comprises 485 amino acids and has 32%sequence identity with human G protein coupled receptor RE2 (SEQ IDNO:80), (FIG. 34). The human sensory GPCR polypeptide of SEQ ID NO:22comprises 316 amino acids and has 47% sequence identity with olfactoryreceptor OR993Gib (SEQ ID NO:81), (FIG. 35). The polynucleotide encodingthe GPCR of SEQ ID NO:22 has been found in cartilage on the basis oftissue expression analysis. The human sensory GPCR polypeptide of SEQ IDNO:23 comprises 311 amino acids and has 76% sequence identity withodorant receptor K11 of mouse (SEQ ID NO:82), (FIG. 36). The humansensory GPCR polypeptide of SEQ ID NO:24 comprises 370 amino acids andhas 78% sequence identity with odorant receptor K4h11 of mouse (SEQ IDNO:83), (FIG. 37). The human sensory GPCR polypeptide of SEQ ID NO:25comprises 255 amino acids and has 43% sequence identity with vomeronasalreceptor V1RC3 of mouse (SEQ ID NO:84), (FIG. 38). The very large humanGPCR polypeptide of SEQ ID NO:26 comprises 295 amino acids and has 30%sequence identity with human very large G-protein coupled receptor-1(SEQ ID NO:85), (FIG. 39). The polynucleotide encoding the GPCR of SEQID NO:26 has been found in brain on the basis of tissue expressionanalysis

[0307] Variants of GPCR polypeptides are also encompassed by the presentinvention. Preferably, a GPCR variant has at least 75 to 80%, morepreferably at least 85 to 90%, and even more preferably at least 90%amino acid sequence identity to a GPCR amino acid sequence disclosedherein, and more preferably, retains at least one biological,immunological, or other functional characteristic or activity of thenon-variant GPCR polypeptide. Most preferred are GPCR variants orsubstantially purified fragments thereof having at least 95% amino acidsequence identity to those of SEQ ID NOs:14-26. Variants of GPCRpolypeptides or substantially purified fragments of the polypeptides canalso include amino acid sequences that differ from any one of the SEQ IDNOs:14-26 amino acid sequences only by conservative substitutions. Theinvention also encompasses polypeptide homologues of any one of aminoacid sequences as set forth in SEQ ID NOs:14-26.

[0308] In another embodiment, the present invention encompassespolynucleotides which encode GPCR polypeptides. Accordingly, any nucleicacid sequence that encodes the amino acid sequence of a GPCR polypeptideof the invention can be used to produce recombinant molecules thatexpress a GPCR protein. More particularly, the invention encompasses theGPCR polynucleotides comprising the nucleic acid sequences of SEQ IDNOs:1-13. The present invention also provides GPCR cDNA clones,specifically clones corresponding to Gene 12, deposited at the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209 on Dec. 22, 2001 and under ATCC Accession No(s). PTA-3949according to the terms of the Budapest Treaty.

[0309] As will be appreciated by the skilled practitioner in the art,the degeneracy of the genetic code results in many nucleotide sequencesthat can encode the described GPCR polypeptides. Some of the sequencesbear minimal or no homology to the nucleotide sequences of any known andnaturally occurring gene. Accordingly, the present inventioncontemplates each and every possible variation of nucleotide sequencethat could be made by selecting combinations based on possible codonchoices. These combinations are made in accordance with the standardtriplet genetic code as applied to the nucleotide sequence of naturallyoccurring GPCR, and all such variations are to be considered as beingspecifically disclosed and able to be understood by the skilledpractitioner.

[0310] Although nucleic acid sequences which encode the GPCRpolypeptides and variants thereof are preferably capable of hybridizingto the nucleotide sequence of the naturally occurring GPCR polypeptideunder appropriately selected conditions of stringency, it may beadvantageous to produce nucleotide sequences encoding GPCR polypeptides,or derivatives thereof, which possess a substantially different codonusage. For example, codons may be selected to increase the rate at whichexpression of the peptide/polypeptide occurs in a particular prokaryoticor eukaryotic host in accordance with the frequency with whichparticular codons are utilized by the host. Another reason forsubstantially altering the nucleotide sequence encoding a GPCRpolypeptide, or its derivatives, without altering the encoded amino acidsequences, includes the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

[0311] The present invention also encompasses production of DNAsequences, or portions thereof, which encode the GPCR polypeptides, orderivatives thereof, entirely by synthetic chemistry. After production,the synthetic sequence may be inserted into any of the many availableexpression vectors and cell systems using reagents that are well knownand practiced by those in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding a GPCR polypeptide,or any fragment thereof.

[0312] In an embodiment of the present invention, a gene delivery vectorcontaining the polynucleotide, or functional fragment thereof isprovided. Preferably, the gene delivery vector contains thepolynucleotide, or functional fragment thereof comprising an isolatedand purified polynucleotide encoding a human GPCR having the sequence asset forth in any one of SEQ ID NOs:1-13.

[0313] It will also be appreciated by those skilled in the pertinent artthat in addition to the primers disclosed in Tables 2 and 3 herein, alonger oligonucleotide probe, or mixtures of probes, for example,degenerate probes, can be used to detect longer, or more complex,nucleic acid sequences, such as, for example, genomic or full lengthDNA. In such cases, the probe may comprise at least 20-300 nucleotides,preferably, at least 30-100 nucleotides, and more preferably, 50-100nucleotides.

[0314] The present invention also provides methods of obtaining the fulllength sequence of the GPCR polypeptides as described herein. In oneinstance, the method of multiplex cloning was devised as a means ofextending large numbers of bioinformatic gene predictions into fulllength sequences by multiplexing probes and cDNA libraries in an effortto minimize the overall effort typically required for cDNA cloning. Themethod relies on the conversion of plasmid-based, directionally clonedcDNA libraries into a population of pure, covalently-closed, circular,single-stranded molecules and long biotinylated DNA oligonucleotideprobes designed from predicted gene sequences.

[0315] Probes and libraries were subjected to solution hybridization ina formamide buffer which has been found to be superior to aqueousbuffers typically used in other biotin/streptavidin cDNA capture methods(i.e., GeneTrapper). The hybridization was performed without priorknowledge of the clones represented in the libraries. Hybridization wasperformed two times. After the first selection, the isolated sequenceswere screened with PCR primers specific for the targeted clones. Thesecond hybridization was carried out with only those oligo probes whosegene-specific PCR assays gave positive results.

[0316] The secondary hybridization serves to ‘normalize’ the selectedlibrary, thereby decreasing the amount of screening needed to identifyparticular clones. The method is robust and sensitive. Typically, dozensof cDNAs are isolated for any one particular gene, thereby increasingthe chances of obtaining a full length cDNA. The entire complexity ofany cDNA library is screened in the solution hybridization process,which is advantageous for finding rare sequences. The procedure isscaleable, with 50 oligonucleotide probes per experiment currently beingused, although this is not to be considered a limiting number.

[0317] Using bioinformatic predicted gene sequence, the following typesof PCR primers and cloning oligos can be designed: A) PCR primer pairsthat reside within a single predicted exon; B) PCR primer pairs thatcross putative exon/intron boundaries; and C) 80mer antisense and senseoligos containing a biotin moiety on the 5′ end. The primer pairs of theA type above are optimized on human genomic DNA; the B type primer pairsare optimized on a mixture of first strand cDNAs made with and withoutreverse transcriptase. Primers will be optimized using mRNA derived fromappropriate tissues sources, for example, brain, lung, uterus,cartilage, and testis poly A+ RNA.

[0318] The information obtained with the B type primers is used toassess those putative expressed sequences which can be experimentallyobserved to have reverse transcriptase-dependent expression. The primerpairs of the A type are less stringent in terms of identifying expressedsequences. However, because they amplify genomic DNA as well as cDNA,their ability to amplify genomic DNA provides for the necessary positivecontrol for the primer pair. Negative results with the B type aresubject to the caveat that the sequence(s) may not be expressed in thetissue first strand that is under examination.

[0319] The biotinylated 80-mer oligonucleotides are added en mass topools of single strand cDNA libraries. Up to 50 probes have beensuccessfully used on pools for 15 different libraries. After the primaryselection is performed, all of the captured DNA is repaired to doublestrand form using the T7 primer for the commercial libraries inpCMVSPORT, and the Sp6 primer for other constructed libraries in pSPORT.The resulting DNA is electroporated into E. coli DH12S and plated onto150 mm plates with nylon filters. The cells are scraped and a frozenstock is made, thereby comprising the primary selected library.

[0320] One-fifth of the library is generally converted into singlestrand form and the DNA is assayed with gene specific primer pairs(GSPs). The next round of solution hybridization capture is carried outwith 80 mer oligos for only those sequences that are positive with thegene-specific-primers. After the second round, the captured singlestrand DNAs are repaired with a pool of GSPs, where only the primercomplementary to polarity of the single-stranded circular DNA is used(i.e., the antisense primer for pCMVSPORT and pSPORT1 and the senseprimer for pSPORT2).

[0321] The resulting colonies are screened by PCR using the GSPs.Typically, greater than 80% of the clones are positive for any givenGSP. The entire 96 well block of clones is subjected to “mini-prep”, asknown in the art, and each of clones is sized by either PCR orrestriction enzyme digestion. A selection of different sized clones foreach targeted sequence is chosen for transposon-hopping and DNAsequencing.

[0322] Preferably, as for established cDNA cloning methods used by theskilled practitioner, the libraries employed are of high quality. Highcomplexity and large average insert size are optimal. High PressureLiquid Chromatography (HPLC) may be employed as a means of fractionatingcDNA for the purpose of constructing libraries.

[0323] Another embodiment of the present invention provides a method ofidentifying full-length genes encoding the disclosed polypeptides. TheGPCR polynucleotides of the present invention, the polynucleotidesencoding the GPCR polypeptides of the present invention, or thepolypeptides encoded by the deposited clone(s) preferably represent thecomplete coding region (i.e., full-length gene).

[0324] Several methods are known in the art for the identification ofthe 5′ or 3′ non-coding and/or coding portions of a given gene. Themethods described herein are exemplary and should not be construed aslimiting the scope of the invention. These methods include, but are notlimited to, filter probing, clone enrichment using specific probes, andprotocols similar or identical to 5′ and 3′ “RACE” protocols that arewell known in the art. For instance, a method similar to 5′ RACE isavailable for generating the missing 5′ end of a desired full-lengthtranscript. (Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684(1993)).

[0325] Briefly, in the RACE method, a specific RNA oligonucleotide isligated to the 5′ ends of a population of RNA presumably containingfull-length gene RNA transcripts. A primer set containing a primerspecific to the ligated RNA oligonucleotide and a primer specific to aknown sequence of the gene of interest is used to PCR amplify the 5′portion of the desired full-length gene. This amplified product may thenbe sequenced and used to generate the full-length gene.

[0326] The above method utilizes total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation is treatedwith phosphatase, if necessary, to eliminate 5′ phosphate groups ondegraded or damaged RNA that may interfere with the later RNA ligasestep. The phosphatase is preferably inactivated and the RNA is treatedwith tobacco acid pyrophosphatase in order to remove the cap structurepresent at the 5′ ends of messenger RNAs. This reaction leaves a 5′phosphate group at the 5′ end of the cap cleaved RNA which can then beligated to an RNA oligonucleotide using T4 RNA ligase.

[0327] The above-described modified RNA preparation is used as atemplate for first strand cDNA synthesis employing a gene specificoligonucleotide. The first strand synthesis reaction is used as atemplate for PCR amplification of the desired 5′ end using a primerspecific to the ligated RNA oligonucleotide and a primer specific to theknown sequence of the gene of interest. The resultant product is thensequenced and analyzed to confirm that the 5′ end sequence belongs tothe desired gene. It may also be advantageous to optimize the RACEprotocol to increase the probability of isolating additional 5′ or 3′coding or non-coding sequences. Various methods of optimizing a RACEprotocol are known in the art; for example, a detailed descriptionsummarizing these methods can be found in B. C. Schaefer, Anal.Biochem., 227:255-273, (1995).

[0328] An alternative method for carrying out 5′ or 3′ RACE for theidentification of coding or non-coding nucleic acid sequences isprovided by Frohman, M. A., et al., Proc. Nat'l. Acad. Sci. USA,85:8998-9002 (1988). Briefly, a cDNA clone missing either the 5′ or 3′end can be reconstructed to include the absent base pairs extending tothe translational start or stop codon, respectively. In some cases,cDNAs are missing the start of translation for an encoded product. Abrief description of a modification of the original 5′ RACE procedure isas follows. Poly A+ or total RNA is reverse transcribed with SuperscriptII (Gibco/BRL) and an antisense or an I complementary primer specific toany one of the cDNA sequences provided as SEQ ID NOS:1-13. The primer isremoved from the reaction with a Microcon Concentrator (Amicon). Thefirst-strand cDNA is then tailed with dATP and terminal deoxynucleotidetransferase (Gibco/BRL). Thus, an anchor sequence is produced which isneeded for PCR amplification. The second strand is synthesized from thedA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), anoligo-dT primer containing three adjacent restriction sites (XhoIJ Sailand ClaI) at the 5′ end and a primer containing just these restrictionsites. This double-stranded cDNA is PCR amplified for 40 cycles with thesame primers, as well as a nested cDNA-specific antisense primer. ThePCR products are size-separated on an ethidium bromide-agarose gel andthe region of gel containing cDNA products having the predicted size ofmissing protein-coding DNA is removed.

[0329] cDNA is purified from the agarose with the Magic PCR Prep kit(Promega), restriction digested with XhoI or SalI, and ligated to aplasmid such as pBluescript SKII (Stratagene) at XhoI and EcoRV sites.This DNA is transformed into bacteria and the plasmid clones sequencedto identify the correct protein-coding inserts. Correct 5′ ends areconfirmed by comparing this sequence with the putatively identifiedhomologue and overlap with the partial cDNA clone. Similar methods knownin the art and/or commercial kits are used to amplify and recover 3′ends.

[0330] Several quality-controlled kits are commercially available forpurchase. Similar reagents and methods to those above are supplied inkit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of fulllength genes. A second kit is available from Clontech which is amodification of a related technique, called single-stranded ligation tosingle-stranded cDNA, (SLIC), developed by Dumas et al., Nucleic AcidsRes., 19:5227-32(1991). The major difference in the latter procedure isthat the RNA is alkaline hydrolyzed after reverse transcription and RNAligase is used to join a restriction site-containing anchor primer tothe first-strand cDNA. This obviates the necessity for the dA-tailingreaction which results in a polyT stretch that can impede sequencing.

[0331] An alternative to generating 5′ or 3′ cDNA from RNA is to usecDNA library double-stranded DNA. An asymmetric PCR-amplified antisensecDNA strand is synthesized with an antisense cDNA-specific primer and aplasmid-anchored primer. These primers are removed and a symmetric PCRreaction is performed with a nested cDNA-specific antisense primer andthe plasmid-anchored primer.

[0332] Also encompassed by the present invention are polynucleotidesequences that are capable of hybridizing to the novel GPCR nucleic acidsequences, as set forth in SEQ ID NOs:1-13, under various conditions ofstringency. Hybridization conditions are typically based on the meltingtemperature (T_(m)) of the nucleic acid binding complex or probe (see,G. M. Wahl and S. L. Berger, 1987; Methods Enzymol., 152:399-407 and A.R. Kimmel, 1987; Methods of Enzymol., 152:507-511), and may be used at adefined stringency. For example, included in the present invention aresequences capable of hybridizing under moderately stringent conditionsto the GPCR sequences of SEQ ID NOs:1-13 and other sequences which aredegenerate to those which encode the novel GPCR polypeptides. Forexample, a non-limiting example of moderate stringency conditionsinclude prewashing solution of 2×SSC, 0.5% SDS, 1.0 mM EDTA, pH 8.0, andhybridization conditions of 50° C., 5×SSC, overnight.

[0333] The nucleic acid sequence encoding the GPCR proteins of thepresent invention may be extended by utilizing a partial nucleotidesequence and employing various methods known in the art to detectupstream sequences such as promoters and regulatory elements. Forexample, one method that can be employed is restriction-site PCR, whichutilizes universal primers to retrieve unknown sequence adjacent to aknown locus (See, e.g., G. Sarkar, 1993, PCR Methods Applic.,2:318-322). In particular, genomic DNA is first amplified in thepresence of a primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

[0334] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region or sequence (T. Triglia etal., 1988, Nucleic Acids Res., 16:8186). The primers may be designedusing OLIGO 4.06 Primer Analysis software (National Biosciences, Inc.,Plymouth, Minn.), or another appropriate program, to be 22-30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68° C.-72° C. Themethod uses several restriction enzymes to generate a suitable fragmentin the known region of a gene. The fragment is then circularized byintramolecular ligation and used as a PCR template.

[0335] Another method which may be used to amplify or extend sequencesis capture PCR which involves PCR amplification of DNA fragmentsadjacent to a known sequence in human and yeast artificial chromosome(YAC) DNA (M. Lagerstrom et al., 1991, PCR Methods Applic., 1:111-119).In this method, multiple restriction enzyme digestions and ligations mayalso be used to place an engineered double-stranded sequence into anunknown portion of the DNA molecule before performing PCR. J. D. Parkeret al. (1991; Nucleic Acids Res., 19:3055-3060) provide another methodwhich may be used to retrieve unknown sequences. Bacterial artificialchromosomes (BACs) are also used for such applications. In addition,PCR, nested primers, and PROMOTERFINDER libraries can be used to “walk”genomic DNA (Clontech, Palo Alto, Calif.). This process avoids the needto screen libraries and is useful in finding intron/exon junctions.

[0336] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are also preferable, since such libraries willcontain more sequences that comprise the 5′ regions of genes. The use ofa randomly primed library may be especially preferable for situations inwhich an oligo d(T) library does not yield a full-length cDNA. Genomiclibraries may be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions.

[0337] The embodiments of the present invention can be practiced usingmethods for DNA sequencing which are well known and generally availablein the art. The methods may employ such enzymes as the Klenow fragmentof DNA polymerase I, SEQUENASE (US Biochemical Corp. Cleveland, Ohio),Taq polymerase (PE Biosystems), thermostable T7 polymerase (AmershamPharmacia Biotech, Piscataway, N.J.), or combinations of recombinantpolymerases and proofreading exonucleases such as the ELONGASEAmplification System marketed by Life Technologies (Gaithersburg, Md.).Preferably, the process is automated with machines such as the HamiltonMicro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200;MJ Research, Watertown, Mass.) and the ABI Catalyst and 373 and 377 DNAsequencers (PE Biosystems). Commercially available capillaryelectrophoresis systems may be used to analyze the size or confirm thenucleotide sequence of sequencing or PCR products. Capillaryelectrophoresis is especially preferable for the sequencing of smallpieces of DNA, which might be present in limited amounts in a particularsample.

[0338] In another embodiment of the present invention, polynucleotidesequences or portions thereof which encode GPCR polypeptides or peptidescan comprise recombinant DNA molecules to direct the expression of GPCRpolypeptide products, peptide fragments, or functional equivalentsthereof, in appropriate host cells. Because of the inherent degeneracyof the genetic code, other DNA sequences, which encode substantially thesame or a functionally equivalent amino acid sequence, may be producedand these sequences may be used to clone and express the GPCR proteinsas described.

[0339] As will be appreciated by those having skill in the art, it maybe advantageous to produce GPCR polypeptide-encoding nucleotidesequences possessing non-naturally occurring codons. For example, codonspreferred by a particular prokaryotic or eukaryotic host can be selectedto increase the rate of protein expression or to produce a recombinantRNA transcript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0340] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterthe GPCR polypeptide-encoding sequences for a variety of reasons,including, but not limited to, alterations which modify the cloning,processing, and/or expression of the gene product. DNA shuffling byrandom fragmentation, PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Forexample, site-directed mutagenesis may be used to insert new restrictionsites, alter glycosylation patterns, change codon preference, producesplice variants, or introduce mutations, and the like.

[0341] In another embodiment of the present invention, natural,modified, or recombinant nucleic acid sequences encoding the GPCRpolypeptides may be ligated to a heterologous sequence to encode afusion (or chimeric or hybrid) protein. For example, a fusion proteincan comprise any one of the amino acid sequences as set forth in SEQ IDNOs:14-26 and an amino acid sequence of an Fc portion (or constantregion) of a human immunoglobulin protein. The fusion protein mayfurther comprise an amino acid sequence that differs from any one of SEQID NOs:14-26 only by conservative substitutions. As another example, toscreen peptide libraries for inhibitors of GPCR activity, it may beuseful to encode a chimeric GPCR protein that can be recognized by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between the GPCR protein-encodingsequence and the heterologous protein sequence, so that the GPCR proteinmay be cleaved and purified away from the heterologous moiety.

[0342] In a further embodiment, sequences encoding the GPCR polypeptidesmay be synthesized in whole, or in part, using chemical methods wellknown in the art (see, for example, M. H. Caruthers et al., 1980, Nucl.Acids Res. Symp. Ser., 215-223 and T. Horn et al., 1980, Nucl. AcidsRes. Symp. Ser., 225-232). Alternatively, the GPCR protein itself, or afragment or portion thereof, may be produced using chemical methods tosynthesize the amino acid sequence of the GPCR polypeptide, or afragment or portion thereof. For example, peptide synthesis can beperformed using various solid-phase techniques (J. Y. Roberge et al.,1995, Science, 269:202-204) and automated synthesis can be achieved, forexample, using the ABI 431A Peptide Synthesizer (PE Biosystems).

[0343] The newly synthesized GPCR polypeptide or peptide can besubstantially purified by preparative high performance liquidchromatography (e.g., T. Creighton, 1983, Proteins, Structures andMolecular Principles, W. H. Freeman and Co., New York, N.Y.), byreverse-phase high performance liquid chromatography (HPLC), or otherpurification methods as known and practiced in the art. The compositionof the synthetic peptides may be confirmed by amino acid analysis orsequencing (e.g., the Edman degradation procedure; Creighton, supra). Inaddition, the amino acid sequence of a GPCR polypeptide, or any portionthereof, can be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

[0344] To express a biologically active GPCR polypeptide or peptide, thenucleotide sequences encoding the GPCR polypeptide, or functionalequivalents, may be inserted into an appropriate expression vector,i.e., a vector, which contains the necessary elements for thetranscription and translation of the inserted coding sequence.

[0345] In one embodiment of the present invention, an expression vectorcontains an isolated and purified polynucleotide sequence as set forthin any one of SEQ ID NOs:1-13, encoding a human GPCR, or a functionalfragment thereof, in which the human GPCR comprises the amino acidsequence as set forth in any one of SEQ ID NOs:14-26. Alternatively, anexpression vector can contain the complement of the aforementioned GPCRnucleic acid sequences.

[0346] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids can be used forthe delivery of nucleotide sequences to a target organ, tissue or cellpopulation. Methods, which are well known to those skilled in the art,may be used to construct expression vectors containing sequencesencoding one or more GPCR polypeptide along with appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Such techniques are described in J.Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, Plainview, N.Y. and in F. M. Ausubel et al., 1989,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y.

[0347] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding the GPCR polypeptides orpeptides. Such expression vector/host systems include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus (CaMV) and tobacco mosaic virus (TMV)), or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems, including mammalian cell systems. The host cell employed is notlimiting to the present invention. Preferably, the host cell of theinvention contains an expression vector comprising an isolated andpurified polynucleotide having a nucleic acid sequence selected from anyone of SEQ ID NOs:1-13 and encoding a human GPCR of this invention, or afunctional fragment thereof, comprising an amino acid sequence as setforth in any one of SEQ ID NOs:14-26.

[0348] Bacterial artificial chromosomes (BACs) may be used to deliverlarger fragments of DNA than can be contained and expressed in a plasmidvector. BACs are vectors used to clone DNA sequences of 100-300 kb, onaverage 150 kb, in size in E. coli cells. BACs are constructed anddelivered via conventional delivery methods (e.g., liposomes,polycationic amino polymers, or vesicles) for therapeutic purposes.

[0349] “Control elements” or “regulatory sequences” are thosenon-translated regions of the vector, e.g., enhancers, promoters, 5′ and3′ untranslated regions, which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Specificinitiation signals may also be used to achieve more efficienttranslation of sequences encoding a GPCR polypeptide. Such signalsinclude the ATG initiation codon and adjacent sequences. In cases wheresequences encoding a GPCR polypeptide, its initiation codon, andupstream sequences are inserted into the appropriate expression vector,no additional transcriptional or translational control signals may beneeded. However, in cases where only a GPCR coding sequence, or afragment thereof, is inserted, exogenous translational control signals,including the ATG initiation codon, are optimally provided. Furthermore,the initiation codon should be in the correct reading frame to insuretranslation of the entire insert. Exogenous translational elements andinitiation codons can be of various origins, both natural and synthetic.The efficiency of expression can be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system that isused, such as those described in the literature (see, e.g., D. Scharf etal., 1994, Results Probl. Cell Differ., 20:125-162).

[0350] In bacterial systems, a number of expression vectors may beselected, depending upon the use intended for the expressed GPCRproduct. For example, when large quantities of expressed protein areneeded for the generation of antibodies, vectors that direct high levelexpression of fusion proteins that can be readily purified may be used.Such vectors include, but are not limited to, the multifunctional E.coli cloning and expression vectors such as BLUESCRIPT (Stratagene), inwhich the sequence encoding the GPCR polypeptide can be ligated into thevector in-frame with sequences for the amino-terminal Met and thesubsequent 7 residues of β-galactosidase, so that a hybrid protein isproduced; pIN vectors (see, G. Van Heeke and S. M. Schuster, 1989, J.Biol. Chem., 264:5503-5509); and the like. pGEX vectors (Promega,Madison, Wis.) can also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can be easily purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems can bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

[0351] In mammalian host cells, a number of viral-based expressionsystems can be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding the GPCR polypeptide may beligated into an adenovirus transcription/translation complex containingthe late promoter and tripartite leader sequence. Insertion into anon-essential E1 or E3 region of the viral genome may be used to obtaina viable virus which is capable of expressing GPCR polypeptide ininfected host cells (J. Logan and T. Shenk, 1984, Proc. Natl. Acad.Sci., 81:3655-3659). In addition, transcription enhancers, such as theRous sarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells. Other expression systems can also be used, suchas, but not limited to yeast, plant, and insect vectors.

[0352] Moreover, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells having specific cellular machinery andcharacteristic mechanisms for such post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138) are available from the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209, and may be chosen to ensure the correct modification andprocessing of the foreign protein.

[0353] Host cells transformed with nucleotide sequences encoding a GPCRprotein, or fragments thereof, may be cultured under conditions suitablefor the expression and recovery of the protein from cell culture. Theprotein produced by a recombinant cell may be secreted or containedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those having skill in the art, expression vectorscontaining polynucleotides which encode a GPCR protein can be designedto contain signal sequences which direct secretion of the GPCR proteinthrough a prokaryotic or eukaryotic cell membrane. Other constructionscan be used to join nucleic acid sequences encoding a GPCR protein to anucleotide sequence encoding a polypeptide domain, which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals; protein A domains that allow purification on immobilizedimmunoglobulin; and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and GPCR protein may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingGPCR and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography) as described by J. Porath et al., 1992, Prot. Exp.Purif., 3:263-281, while the enterokinase cleavage site provides a meansfor purifying from the fusion protein. For a discussion of suitablevectors for fusion protein production, see D. J. Kroll et al., 1993; DNACell Biol., 12:441-453.

[0354] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theHerpes Simplex Virus thymidine kinase (HSV TK), (M. Wigler et al., 1977,Cell, 11:223-32) and adenine phosphoribosyltransferase (I. Lowy et al.,1980, Cell, 22:817-23) genes which can be employed in tk⁻ or aprt⁻cells, respectively. Also, anti-metabolite, antibiotic or herbicideresistance can be used as the basis for selection; for example, dhfr,which confers resistance to methotrexate (M. Wigler et al., 1980, Proc.Natl. Acad. Sci., 77:3567-70); npt, which confers resistance to theaminoglycosides neomycin and G-418 (F. Colbere-Garapin et al., 1981, J.Mol. Biol., 150:1-14); and als or pat, which confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively(Murry, supra). Additional selectable genes have been described, forexample, trpB, which allows cells to utilize indole in place oftryptophan, or hisD, which allows cells to utilize histinol in place ofhistidine (S. C. Hartman and R. C. Mulligan, 1988, Proc. Natl. Acad.Sci., 85:8047-51). Recently, the use of visible markers has gainedpopularity with such markers as the anthocyanins, β-glucuronidase andits substrate GUS, and luciferase and its substrate luciferin, which arewidely used not only to identify transformants, but also to quantify theamount of transient or stable protein expression that is attributable toa specific vector system (C. A. Rhodes et al., 1995, Methods Mol. Biol.,55:121-131).

[0355] Although the presence or absence of marker gene expressionsuggests that the gene of interest is also present, the presence andexpression of the desired gene of interest may need to be confirmed. Forexample, if the nucleic acid sequence encoding a GPCR polypeptide isinserted within a marker gene sequence, recombinant cells containingpolynucleotide sequence encoding the GPCR polypeptide can be identifiedby the absence of marker gene function. Alternatively, a marker gene canbe placed in tandem with a sequence encoding a GPCR polypeptide underthe control of a single promoter. Expression of the marker gene inresponse to induction or selection typically indicates co-expression ofthe tandem gene.

[0356] A wide variety of labels and conjugation techniques are known andemployed by those skilled in the art and may be used in various nucleicacid and amino acid assays. Means for producing labeled hybridization orPCR probes for detecting sequences related to polynucleotides encoding aGPCR polypeptide include oligo-labeling, nick translation, end-labeling,or PCR amplification using a labeled nucleotide. Alternatively, thesequences encoding a GPCR polypeptide of this invention, or any portionor fragment thereof, can be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase, such as T7, T3, or SP(6) and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (e.g., Amersham Pharmacia Biotech, Promegaand U.S. Biochemical Corp.). Suitable reporter molecules or labels whichcan be used include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents, as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

[0357] Alternatively, host cells which contain the nucleic acid sequencecoding for a GPCR polypeptide of the invention and which express theGPCR polypeptide product may be identified by a variety of proceduresknown to those having skill in the art. These procedures include, butare not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques, including membrane, solution, orchip based technologies, for the detection and/or quantification ofnucleic acid or protein.

[0358] The presence of polynucleotide sequences encoding GPCRpolypeptides can be detected by DNA-DNA or DNA-RNA hybridization, or byamplification using probes, portions, or fragments of polynucleotidesencoding a GPCR polypeptide. Nucleic acid amplification based assaysinvolve the use of oligonucleotides or oligomers based on the nucleicacid sequences encoding a GPCR polypeptide to detect transformantscontaining DNA or RNA encoding GPCR polypeptide.

[0359] In addition to recombinant production, fragments of GPCRpolypeptides may be produced by direct peptide synthesis using solidphase techniques (J. Merrifield, 1963, J. Am. Chem. Soc., 85:2149-2154).Protein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, using ABI431A Peptide Synthesizer (PE Biosystems). Various fragments of the GPCRpolypeptides can be chemically synthesized separately and then combinedusing chemical methods to produce the full length molecule.

[0360] Diagnostic Assays

[0361] In another embodiment of the present invention, antibodies whichspecifically bind to a GPCR polypeptide may be used for the diagnosis ofconditions or diseases characterized by expression (or overexpression)of the GPCR polynucleotide or polypeptide, or in assays to monitorpatients being treated with one or more of the GPCR polypeptides, oragonists, antagonists, or inhibitors of the novel GPCRs. The antibodiesuseful for diagnostic purposes can be prepared in the same manner asthose described herein for use in therapeutic methods. Diagnostic assaysfor the GPCR polypeptides include methods which utilize the antibody anda label to detect the protein in human body fluids or extracts of cellsor tissues. The antibodies may be used with or without modification, andmay be labeled by joining them, either covalently or non-covalently,with a reporter molecule. A wide variety of reporter molecules known tothose in the art may be used, several of which are described herein.

[0362] Another embodiment of the present invention contemplates a methodof detecting a GPCR homologue, or an antibody-reactive fragment thereof,in a sample. The method comprises a) contacting the sample with anantibody specific for a GPCR polypeptide of the present invention, or anantigenic fragment thereof, under conditions in which anantigen-antibody complex can form between the antibody and thepolypeptide or antigenic fragment thereof in the sample; and b)detecting the antigen-antibody complex formed in step a), whereindetection of the complex indicates the presence of the GPCR polypeptide,or an antigenic fragment thereof, in the sample.

[0363] Several assay protocols including enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RIA), and fluorescence activated cellsorting (FACS) for measuring GPCR polypeptide are known in the art andprovide a basis for diagnosing altered or abnormal levels of GPCRpolypeptide expression. Normal or standard values for GPCR polypeptideexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody tothe GPCR polypeptide under conditions suitable for complex formation.The amount of standard complex formation may be quantified by variousmethods; photometric means are preferred. Quantities of GPCR polypeptideexpressed in a subject or test sample, control sample, and diseasesamples from biopsied tissues are compared with the standard values.Deviation between standard and subject values establishes the parametersfor diagnosing disease.

[0364] A variety of protocols for detecting and measuring the expressionof GPCR polypeptide using either polyclonal or monoclonal antibodiesspecific for the polypeptide, or epitopic portions thereof, are knownand practiced in the art. Examples include enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RIA), and fluorescence activated cellsorting (FACS). A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive with two non-interfering epitopes on aGPCR polypeptide is preferred, but a competitive binding assay may alsobe employed. These and other assays are described in the art asrepresented by the publication of R. Hampton et al., 1990; SerologicalMethods, a Laboratory Manual, APS Press, St Paul, Minn. and D. E. Maddoxet al., 1983; J. Exp. Med., 158:1211-1216).

[0365] Uses for Antibodies Directed Against Polypeptides of theInvention

[0366] The antibodies of the present invention have various utilities.For example, such antibodies may be used in diagnostic assays to detectthe presence or quantification of the polypeptides of the invention in asample. Such a diagnostic assay may be comprised of at least two steps.The first, subjecting a sample with the antibody, wherein the sample isa tissue (e.g., human, animal, etc.), biological fluid (e.g., blood,urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract(e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g.,See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or achromatography column, etc. And a second step involving thequantification of antibody bound to the substrate. Alternatively, themethod may additionally involve a first step of attaching the antibody,either covalently, electrostatically, or reversibly, to a solid support,and a second step of subjecting the bound antibody to the sample, asdefined above and elsewhere herein.

[0367] Various diagnostic assay techniques are known in the art, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc., (1987), pp147-158). The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase, green fluorescent protein, or horseradishperoxidase. Any method known in the art for conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem.,13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219(1981); andNygren, J. Histochem. And Cytochem., 30:407 (1982).

[0368] Antibodies directed against the polypeptides of the presentinvention are useful for the affinity purification of such polypeptidesfrom recombinant cell culture or natural sources. In this process, theantibodies against a particular polypeptide are immobilized on asuitable support, such as a Sephadex resin or filter paper, usingmethods well known in the art. The immobilized antibody then iscontacted with a sample containing the polypeptides to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except for thedesired polypeptides, which are bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the desired polypeptide from the antibody.

[0369] Immunophenotyping

[0370] The antibodies of the invention may be utilized forimmunophenotyping of cell lines and biological samples. The translationproduct of the gene of the present invention may be useful as a cellspecific marker, or more specifically as a cellular marker that isdifferentially expressed at various stages of differentiation and/ormaturation of particular cell types. Monoclonal antibodies directedagainst a specific epitope, or combination of epitopes, will allow forthe screening of cellular populations expressing the marker. Varioustechniques can be utilized using monoclonal antibodies to screen forcellular populations expressing the marker(s), and include magneticseparation using antibody-coated magnetic beads, “panning” with antibodyattached to a solid matrix (i.e., plate), and flow cytometry (See, e.g.,U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

[0371] These techniques allow for the screening of particularpopulations of cells, such as might be found with hematologicalmalignancies (i.e. minimal residual disease (MRD) in acute leukemicpatients) and “non-self” cells in transplantations to preventGraft-versus-Host Disease (GVHD). Alternatively, these techniques allowfor the screening of hematopoietic stem and progenitor cells capable ofundergoing proliferation and/or differentiation, as might be found inhuman umbilical cord blood.

[0372] Assays for Antibody Binding

[0373] The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

[0374] Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1-4 hours) at 4° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 4° C., washing the beads inlysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0375] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., 32P or 125I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0376] ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

[0377] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., 3H or 125I) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the antigen is incubated with antibody of interest conjugated to alabeled compound (e.g., 3H or 125I) in the presence of increasingamounts of an unlabeled second antibody.

[0378] Therapeutic Uses Of Antibodies

[0379] The present invention is further directed to antibody-basedtherapies which involve administering antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, patient fortreating one or more of the disclosed diseases, disorders, orconditions. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (including fragments, analogsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

[0380] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0381] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic-growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

[0382] The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

[0383] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10−2 M,10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M,10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M,10−10 M, 5×10−11 M, 10-11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M,5×10−14 M, 10−14 M, 5×10−15 M, and 10−15 M.

[0384] Antibodies directed against polypeptides of the present inventionare useful for inhibiting allergic reactions in animals. For example, byadministering a therapeutically acceptable dose of an antibody, orantibodies, of the present invention, or a cocktail of the presentantibodies, or in combination with other antibodies of varying sources,the animal may not elicit an allergic response to antigens.

[0385] Likewise, one could envision cloning the gene encoding anantibody directed against a polypeptide of the present invention, saidpolypeptide having the potential to elicit an allergic and/or immuneresponse in an organism, and transforming the organism with saidantibody gene such that it is expressed (e.g., constitutively,inducibly, etc.) in the organism. Thus, the organism would effectivelybecome resistant to an allergic response resulting from the ingestion orpresence of such an immune/allergic reactive polypeptide. Moreover, sucha use of the antibodies of the present invention may have particularutility in preventing and/or ameliorating autoimmune diseases and/ordisorders, as such conditions are typically a result of antibodies beingdirected against endogenous proteins. For example, in the instance wherethe polypeptide of the present invention is responsible for modulatingthe immune response to auto-antigens, transforming the organism and/orindividual with a construct comprising any of the promoters disclosedherein or otherwise known in the art, in addition, to a polynucleotideencoding the antibody directed against the polypeptide of the presentinvention could effective inhibit the organisms immune system fromeliciting an immune response to the auto-antigen(s). Detaileddescriptions of therapeutic and/or gene therapy applications of thepresent invention are provided elsewhere herein.

[0386] Alternatively, antibodies of the present invention could beproduced in a plant (e.g., cloning the gene of the antibody directedagainst a polypeptide of the present invention, and transforming a plantwith a suitable vector comprising said gene for constitutive expressionof the antibody within the plant), and the plant subsequently ingestedby an animal, thereby conferring temporary immunity to the animal forthe specific antigen the antibody is directed towards (See, for example,U.S. Pat. Nos. 5,914,123 and 6,034,298).

[0387] In another embodiment, antibodies of the present invention,preferably polyclonal antibodies, more preferably monoclonal antibodies,and most preferably single-chain antibodies, can be used as a means ofinhibiting gene expression of a particular gene, or genes, in a human,mammal, and/or other organism. See, for example, InternationalPublication Number WO 00/05391, published Feb. 3, 2000, to DowAgrosciences LLC. The application of such methods for the antibodies ofthe present invention are known in the art, and are more particularlydescribed elsewhere herein.

[0388] In yet another embodiment, antibodies of the present inventionmay be useful for multimerizing the polypeptides of the presentinvention. For example, certain proteins may confer enhanced biologicalactivity when present in a multimeric state (i.e., such enhancedactivity may be due to the increased effective concentration of suchproteins whereby more protein is available in a localized location).

[0389] Antibody-Based Gene Therapy

[0390] In a specific embodiment, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of a polypeptide of the invention,by way of gene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

[0391] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0392] For general reviews of the methods of gene therapy, see Goldspielet al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596(1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0393] In a preferred aspect, the compound comprises nucleic acidsequences encoding an antibody, said nucleic acid sequences being partof expression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

[0394] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0395] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

[0396] In a specific embodiment, viral vectors that contains nucleicacid sequences encoding an antibody of the invention are used. Forexample, a retroviral vector can be used (see Miller et al., Meth.Enzymol. 217:581-599 (1993)). These retroviral vectors contain thecomponents necessary for the correct packaging of the viral genome andintegration into the host cell DNA. The-nucleic acid sequences encodingthe antibody to be used in gene therapy are cloned into one or morevectors, which facilitates delivery of the gene into a patient. Moredetail about retroviral vectors can be found in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993).

[0397] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

[0398] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300(1993); U.S. Pat. No. 5,436,146).

[0399] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0400] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0401] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0402] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0403] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0404] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an antibody are introduced intothe cells such that they are expressible by the cells or their progeny,and the recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

[0405] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription. Demonstration of Therapeutic or ProphylacticActivity

[0406] The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

[0407] Therapeutic/Prophylactic Administration and Compositions

[0408] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount of acompound or pharmaceutical composition of the invention, preferably anantibody of the invention. In a preferred aspect, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human.

[0409] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0410] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

[0411] In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

[0412] In another embodiment, the compound or composition can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapyof-Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327;see generally ibid.)

[0413] In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

[0414] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0415] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0416] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0417] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0418] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0419] The amount of the compound of the invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0420] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

[0421] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

[0422] Diagnosis and Imaging With Antibodies

[0423] Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

[0424] The invention provides a diagnostic assay for diagnosing adisorder, comprising (a) assaying the expression of the polypeptide ofinterest in cells or body fluid of an individual using one or moreantibodies specific to the polypeptide interest and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a particulardisorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0425] Antibodies of the invention can be used to assay protein levelsin a biological sample using classical immunohistological methods knownto those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0426] One aspect of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled molecule in the subject, such that detection of labeled moleculeabove the background level indicates that-the subject has a particulardisease or disorder associated with aberrant expression of thepolypeptide of interest. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

[0427] It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietyneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of 99 mTc. The labeledantibody or antibody fragment will then preferentially accumulate at thelocation of cells which contain the specific protein. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

[0428] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0429] In an embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisease, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

[0430] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

[0431] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

[0432] Kits

[0433] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

[0434] In another specific embodiment of the present invention, the kitis a diagnostic kit for use in screening serum containing antibodiesspecific against proliferative and/or cancerous polynucleotides andpolypeptides. Such a kit may include a control antibody that does notreact with the polypeptide of interest. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope whichis specifically immunoreactive with at least one anti-polypeptideantigen antibody. Further, such a kit includes means for detecting thebinding of said antibody to the antigen (e.g., the antibody may beconjugated to a fluorescent compound such as fluorescein or rhodaminewhich can be detected by flow cytometry). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedpolypeptide antigen. The polypeptide antigen of the kit may also beattached to a solid support.

[0435] In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the polypeptide antigen can be detected by binding of thesaid reporter-labeled antibody.

[0436] In an additional embodiment, the invention includes a diagnostickit for use in screening serum containing antigens of the polypeptide ofthe invention. The diagnostic kit includes a substantially isolatedantibody specifically immunoreactive with polypeptide or polynucleotideantigens, and means for detecting the binding of the polynucleotide orpolypeptide antigen to the antibody. In one embodiment, the antibody isattached to a solid support. In a specific embodiment, the antibody maybe a monoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

[0437] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound antigen obtained by themethods of the present invention. After binding with specific antigenantibody to the reagent and removing unbound serum components bywashing, the reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-antigen antibody on the solid support. The reagent is againwashed to remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

[0438] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

[0439] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

[0440] In another embodiment of the present invention, a method of usinga GPCR-encoding polynucleotide sequence to purify a molecule or compoundin a sample, wherein the molecule or compound specifically binds to thepolynucleotide, is contemplated. The method comprises: a) combining aGPCR-encoding polynucleotide of the invention with a sample undergoingtesting to determine if the sample contains the molecule or compound,under conditions to allow specific binding; b) detecting specificbinding between the GPCR-encoding polynucleotide and the molecule orcompound, if present; c) recovering the bound polynucleotide; and d)separating the polynucleotide from the molecule or compound, therebyobtaining a purified molecule or compound.

[0441] This invention also relates to a method of using GPCRpolynucleotides as diagnostic reagents. For example, the detection of amutated form of the GPCR gene associated with a dysfunction can providea diagnostic tool that can add to or define diagnosis of a disease orsusceptibility to a disease which results from under-expression,over-expression, or altered expression of GPCRs. Individuals carryingmutations in the GPCR gene may be detected at the DNA level by a varietyof techniques.

[0442] Nucleic acids for diagnosis may be obtained from various sourcesof a subject, for example, from cells, tissue, blood, urine, saliva,tissue biopsy or autopsy material. Genomic DNA may be used directly fordetection or may be amplified by using PCR or other amplificationtechniques prior to analysis. RNA or cDNA may also be used in similarfashion. Deletions and insertions in GPCR-encoding polynucleotide can bedetected by a change in size of the amplified product compared with thatof the normal genotype. Hybridizing amplified DNA to labeled GPCRpolynucleotide sequences can identify point mutations. Perfectly matchedsequences can be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in electrophoreticmobility of DNA fragments in gels, with or without denaturing agents, orby direct DNA sequencing. See, for example, Myers et al., Science (1985)230:1242. Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method. (See Cotton et al., Proc. Natl. Acad. Sci.,USA (1985) 85:43297-4401).

[0443] In another embodiment, an array of oligonucleotide probescomprising GPCR nucleotide sequence or fragments thereof can beconstructed to conduct efficient screening of, for example, geneticmutations. Array technology methods are well known, have generalapplicability and can be used to address a variety of questions inmolecular genetics, including gene expression, genetic linkage, andgenetic variability (see for example: M. Chee et al., Science,274:610-613, 1996).

[0444] Yet another aspect of the present invention involves a method ofscreening a library of molecules or compounds with a GPCR-encodingpolynucleotide to identify at least one molecule or compound thereinwhich specifically binds to the GPCR polynucleotide sequence. Such amethod includes a) combining a GPCR-encoding polynucleotide of thepresent invention with a library of molecules or compounds underconditions to allow specific binding; and b) detecting specific binding,thereby identifying a molecule or compound, which specifically binds toa GPCR-encoding polynucleotide sequence, wherein the library is selectedfrom DNA molecules, RNA molecules, artificial chromosome constructions,PNAs, peptides and proteins.

[0445] The present invention provides diagnostic assays for determiningor monitoring through detection of a mutation in a GPCR gene(polynucleotide) described herein susceptibility to the followingconditions, diseases, or disorders: cancers; anorexia; bulimia; asthma;Parkinson's disease; acute heart failure; hypotension; hypertension;urinary retention; osteoporosis; angina pectoris; myocardial infarction;ulcers; asthma; allergies; benign prostatic hypertrophy; and psychoticand neurological disorders, including anxiety, headache, migraine,schizophrenia, manic depression, delirium, dementia, severe mentalretardation and dyskinesias, such as Huntington's disease or Gilles dela Tourette's syndrome.

[0446] In addition, such diseases, disorder, or conditions, can bediagnosed by methods of determining from a sample derived from a subjecthaving an abnormally decreased or increased level of GPCR polypeptide orGPCR mRNA. Decreased or increased expression can be measured at the RNAlevel using any of the methods well known in the art for thequantification of polynucleotides, such as, for example, PCR, RT-PCR,RNase protection, Northern blotting and other hybridization methods.Assay techniques that can be used to determine levels of a protein, suchas a GPCR in a sample derived from a host are well known to those ofskill in the art. Such assay methods include, without limitation,radioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays.

[0447] In another of its aspects, this invention relates to a kit fordetecting and diagnosing a GPCR-associated disease or susceptibility tosuch a disease, which comprises a GPCR polynucleotide, preferably thenucleotide sequence of SEQ ID NOs:1-13, or a fragment thereof; or anucleotide sequence complementary to the GPCR polynucleotide of SEQ IDNOs:1-13; or a GPCR polypeptide, preferably the polypeptide of SEQ IDNOs:14-26, or a fragment thereof; or an antibody to the GPCRpolypeptide, preferably to the polypeptide of SEQ ID NOs:14-26, anepitope-containing portion thereof, or combinations of the foregoing. Itwill be appreciated that in any such kit, any of the previouslymentioned components may comprise a substantial component. Alsopreferably included are instructions for use.

[0448] The GPCR polynucleotides which may be used in the diagnosticassays according to the present invention include oligonucleotidesequences, complementary RNA and DNA molecules, and PNAs. Thepolynucleotides may be used to detect and quantify GPCR-encoding nucleicacid expression in biopsied tissues in which expression (or under- orover-expression) of the GPCR polynucleotide may be determined, as wellas correlated with disease. The diagnostic assays may be used todistinguish between the absence of GPCR, the presence of GPCR, or theexcess expression of GPCR, and to monitor the regulation of GPCRpolynucleotide levels during therapeutic treatment or intervention.

[0449] In a related aspect, hybridization with PCR probes which arecapable of detecting polynucleotide sequences, including genomicsequences, encoding a GPCR polypeptide according to the presentinvention, or closely related molecules, may be used to identify nucleicacid sequences which encode a GPCR polypeptide. The specificity of theprobe, whether it is made from a highly specific region, for example,about 8 to 10 contiguous nucleotides in the 5′ regulatory region, or aless specific region, for example, especially in the 3′ coding region,and the stringency of the hybridization or amplification (maximal, high,intermediate, or low) will determine whether the probe identifies onlynaturally occurring sequences encoding GPCR polypeptide, allelesthereof, or related sequences.

[0450] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides encodingthe GPCR polypeptide. The hybridization probes or primers of thisinvention may be DNA or RNA and may be derived from the nucleotidesequences of SEQ ID NOs:1-13, or as listed in Tables 2 and 3, or may bederived from genomic sequences including promoter, enhancer elements,and introns of the naturally occurring GPCR protein, wherein the probesor primers comprise a polynucleotide sequence capable of hybridizingwith a polynucleotide of SEQ ID NOs:1-13, under low, moderate, or highstringency conditions.

[0451] Methods for producing specific hybridization probes for DNAencoding the GPCR polypeptides include the cloning of a nucleic acidsequence that encodes the GPCR polypeptide, or GPCR derivatives, intovectors for the production of mRNA probes. Such vectors are known in theart, or are commercially available, and may be used to synthesize RNAprobes in vitro by means of the addition of the appropriate RNApolymerases and the appropriate labeled nucleotides. Hybridizationprobes may be labeled by a variety of detector/reporter groups,including, but not limited to, radionuclides such as ³²P or ³⁵S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0452] The polynucleotide sequences encoding the GPCR polypeptides ofthis invention, or fragments thereof, may be used for the diagnosis ofdisorders associated with expression of GPCRs. The polynucleotidesequence encoding the GPCR polypeptide may be used in Southern orNorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dipstick, pin, ELISA or chip assays utilizingfluids or tissues from patient biopsies to detect the status of, forexample, levels of, or overexpression of, a GPCR, or to detect alteredGPCR expression or levels. Such qualitative or quantitative methods arecommonly practiced in the art.

[0453] In a particular aspect, a nucleotide sequence encoding a GPCRpolypeptide as described herein may be useful in assays that detectactivation or induction of various neoplasms, cancers, or otherGPCR-related diseases, disorders, or conditions. The nucleotide sequenceencoding a GPCR polypeptide may be labeled by standard methods, andadded to a fluid or tissue sample from a patient, under conditionssuitable for the formation of hybridization complexes. After a suitableincubation period, the sample is washed and the signal is quantified andcompared with a standard value. If the amount of signal in the biopsiedor extracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequence has hybridized with nucleotidesequence present in the sample, and the presence of altered levels ofnucleotide sequence encoding the GPCR polypeptide in the sampleindicates the presence of the associated disease. Such assays may alsobe used to evaluate the efficacy of a particular therapeutic treatmentregimen in animal studies, in clinical trials, or in monitoring thetreatment or responsiveness of an individual patient.

[0454] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in a normal individual. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0455] With respect to tumors or cancer, the presence of an abnormalamount or level of a GPCR transcript in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health practitioners to employ preventative measuresor aggressive treatment earlier, thereby preventing the development orfurther progression of the tumor or cancer.

[0456] Additional diagnostic uses for oligonucleotides designed from thenucleic acid sequences encoding the novel GPCR polypeptides of thisinvention can involve the use of PCR. Such oligomers may be chemicallysynthesized, generated enzymatically, or produced from a recombinantsource. Oligomers will preferably comprise two nucleotide sequences: onewith sense orientation (5′→3′) and another with antisense orientation(3′→5′), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantification of closelyrelated DNA or RNA sequences.

[0457] Methods suitable for quantifying the expression of GPCR includeradiolabeling or biotinylating nucleotides, co-amplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (P. C. Melby et al., 1993, J. Immunol. Methods,159:235-244; and C. Duplaa et al., 1993, Anal. Biochem., 229-236). Thespeed of quantifying multiple samples may be accelerated by running theassay in an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or calorimetric responsegives rapid quantification.

[0458] In one embodiment of the invention, a compound to be tested canbe radioactively, calorimetrically or fluorimetrically labeled usingmethods well known in the art and incubated with the GPCR for testing.After incubation, it is determined whether the test compound is bound tothe GPCR polypeptide. If so, the compound is to be considered apotential agonist or antagonist. Functional assays are performed todetermine whether the receptor activity is activated (or enhanced orincreased) or inhibited (or decreased or reduced). These assays include,but are not limited to, cell cycle analysis and in vivo tumor formationassays. Responses can also be measured in cells expressing the receptorusing signal transduction systems including, but not limited to, proteinphosphorylation, adenylate cyclase activity, phosphoinositidehydrolysis, guanylate cyclase activity, ion fluxes (i.e. calcium) and pHchanges. These types of responses can either be present in the host cellor introduced into the host cell along with the receptor.

[0459] The present invention further embraces a method of screening forcandidate compounds capable of modulating the activity of aGPCR-encoding polypeptide. Such a method comprises a) contacting a testcompound with a cell or tissue expressing a GPCR polypeptide of theinvention (e.g., recombinant expression); and b) selecting as candidatemodulating compounds those test compounds that modulate activity of theGPCR polypeptide. Those candidate compounds which modulate GPCR activityare preferably agonists or antagonists, more preferably antagonists ofGPCR activity.

[0460] The present invention encompasses the identification of compoundsand drugs which stimulate Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or13 on the one hand (i.e., agonists) and which inhibit the function ofGene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 on the other hand(i.e., antagonists). In general, such screening procedures involveproviding appropriate cells which express the receptor polypeptide ofthe present invention on the surface thereof. Such cells may include,for example, cells from mammals, yeast, Drosophila or E. coli. In apreferred embodimenta, a polynucleotide encoding the receptor of thepresent invention may be employed to transfect cells to thereby expressthe Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 polypeptide. Theexpressed receptor may then be contacted with a test compound to observebinding, stimulation or inhibition of a functional response.

[0461] One such screening procedure involves the use of melanophoreswhich are transfected to express the Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or 13 polypeptide of the present invention. Such a screeningtechnique is described in PCT WO 92/01810, published Feb. 6, 1992. Suchan assay may be employed to screen for a compound which inhibitsactivation of the receptor polypeptide of the present invention bycontacting the melanophore cells which encode the receptor with both thereceptor ligand, such as LPA, and a compound to be screened. Inhibitionof the signal generated by the ligand indicates that a compound is apotential antagonist for the receptor, i.e., inhibits activation of thereceptor.

[0462] The technique may also be employed for screening of compoundswhich activate the receptor by contacting such cells with compounds tobe screened and determining whether such compound generates a signal,i.e., activates the receptor. Other screening techniques include the useof cells which express the Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,or 13 polypeptide (for example, transfected CHO cells) in a system whichmeasures extracellular pH changes caused by receptor activation. In thistechnique, compounds may be contacted with cells expressing the receptorpolypeptide of the present invention. A second messenger response, e.g.,signal transduction or pH changes, is then measured to determine whetherthe potential compound activates or inhibits the receptor.

[0463] Another screening technique involves expressing the Gene 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 polypeptide in which the receptor islinked to phospholipase C or D. Representative examples of such cellsinclude, but are not limited to, endothelial cells, smooth muscle cells,and embryonic kidney cells. The screening may be accomplished ashereinabove described by detecting activation of the receptor orinhibition of activation of the receptor from the phospholipase secondsignal.

[0464] Another method involves screening for compounds which areantagonists or agonists by determining inhibition of binding of labeledligand, such as LPA, to cells which have the receptor on the surfacethereof, or cell membranes containing the receptor. Such a methodinvolves transfecting a cell (such as eukaryotic cell) with DNA encodingthe Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 polypeptide suchthat the cell expresses the receptor on its surface. The cell is thencontacted with a potential antagonist or agonist in the presence of alabeled form of a ligand, such as LPA. The ligand can be labeled, e.g.,by radioactivity. The amount of labeled ligand bound to the receptors ismeasured, e.g., by measuring radioactivity associated with transfectedcells or membrane from these cells. If the compound binds to thereceptor, the binding of labeled ligand to the receptor is inhibited asdetermined by a reduction of labeled ligand which binds to thereceptors. This method is called binding assay.

[0465] Another screening procedure involves the use of mammalian cells(CHO, HEK 293, Xenopus Oocytes, RBL-2H3, etc) which are transfected toexpress the receptor of interest. The cells are loaded with an indicatordye that produces a fluorescent signal when bound to calcium, and thecells are contacted with a test substance and a receptor agonist, suchas LPA. Any change in fluorescent signal is measured over a definedperiod of time using, for example, a fluorescence spectrophotometer or afluorescence imaging plate reader. A change in the fluorescence signalpattern generated by the ligand indicates that a compound is a potentialantagonist or agonist for the receptor.

[0466] Another screening procedure involves use of mammalian cells (CHO,HEK293, Xenopus Oocytes, RBL-2H3, etc.) which are transfected to expressthe receptor of interest, and which are also transfected with a reportergene construct that is coupled to activation of the receptor (forexample, luciferase or beta-galactosidase behind an appropriatepromoter). The cells are contacted with a test substance and thereceptor agonist (ligand), such as LPA, and the signal produced by thereporter gene is measured after a defined period of time. The signal canbe measured using a luminometer, spectrophotometer, fluorimeter, orother such instrument appropriate for the specific reporter constructused. Change of the signal generated by the ligand indicates that acompound is a potential antagonist or agonist for the receptor.

[0467] Another screening technique for antagonists or agonits involvesintroducing RNA encoding-the Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,or 13 polypeptide into Xenopus oocytes (or CHO, HEK 293, RBL-2H3, etc.)to transiently or stably express the receptor. The receptor oocytes arethen contacted with the receptor ligand, such as LPA, and a compound tobe screened. Inhibition or activation of the receptor is then determinedby detection of a signal, such as, cAMP, calcium, proton, or other ions.

[0468] Another method involves screening for Gene 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, or 13 polypeptide inhibitors by determining inhibitionor stimulation of Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13polypeptide-mediated cAMP and/or adenylate cyclase accumulation ordimunition. Such a method involves transiently or stably transfecting aeukaryotic cell with Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13polypeptide receptor to express the receptor on the cell surface.

[0469] The cell is then exposed to potential antagonists or agonists inthe presence of Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13polypeptide ligand, such as LPA. The changes in levels of cAMP is thenmeasured over a defined period of time, for example, by radio-immuno orprotein binding assays (for example using Flashplates or a scintillationproximity assay). Changes in cAMP levels can also be determined bydirectly measuring the activity of the enzyme, adenylyl cyclase, inbroken cell preparations. If the potential antagonist or agonist bindsthe receptor, and thus inhibits Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or 13 polypeptide-ligand binding, the levels of Gene 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, or 13 polypeptide-mediated cAMP, or adenylatecyclase activity, will be reduced or increased.

[0470] One preferred screening method involves co-transfecting HEK-293cells with a mammalian expression plasmid encoding a G-protein coupledreceptor (GPCR), such as Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or13, along with a mixture comprised of mammalian expression plasmidscDNAs encoding GU15 (Wilkie T. M. et al Proc Natl Acad Sci USA 1991 88:10049-10053), GU16 (Amatruda T. T. et al Proc Natl Acad Sci USA 1991 8:5587-5591, and three chimeric G-proteins refered to as Gqi5, Gqs5, andGqo5 (Conklin B R et al Nature 1993 363: 274-276, Conklin B. R. et alMol Pharmacol 1996 50: 885-890). Following a 24 h incubation thetrasfected HEK-293 cells are plated into poly-D-lysine coated 96 wellblack/clear plates (Becton Dickinson, Bedford, Mass.).

[0471] The cells are assayed on FLIPR (Fluorescent Imaging Plate Reader,Molecular Devices, Sunnyvale, Calif.) for a calcium mobilizationresponse following addition of test ligands. Upon identification of aligand which stimulates calcium mobilization in HEK-293 cells expressinga given GPCR and the G-protein mixtures, subsequent experiments areperformed to determine which, if any, G-protein is required for thefunctional response. HEK-293 cells are then transfected with the testGPCR, or co-transfected with the test GPCR and GO15, GD16, GqiS, Gqs5,or Gqo5. If the GPCR requires the presence of one of the G-proteins forfunctional expression in HEK-293 cells, all subsequent experiments areperformed with HEK-293 cell cotransfected with the GPCR and theG-protein which gives the best response. Alternatively, the receptor canbe expressed in a different cell line, for example RBL-2H3, withoutadditional Gproteins.

[0472] Another screening method for agonists and antagonists relies onthe endogenous pheromone response pathway in the yeast, Saccharomycescerevisiae. Heterothallic strains of yeast can exist in two mitoticallystable haploid mating types, MATa and MATa. Each cell type secretes asmall peptide hormone that binds to a G-protein coupled receptor onopposite mating type cells which triggers a MAP kinase cascade leadingto G1 arrest as a prelude to cell fusion.

[0473] Genetic alteration of certain genes in the pheromone responsepathway can alter the normal response to pheromone, and heterologousexpression and coupling of human G-protein coupled receptors andhumanized G-protein subunits in yeast cells devoid of endogenouspheromone receptors can be linked to downstream signaling pathways andreporter genes (e.g., U.S. Pat. Nos. 5,063,154; 5,482,835; 5,691,188).Such genetic alterations include, but are not limited to, (i) deletionof the STE2 or STE3 gene encoding the endogenous G-protein coupledpheromone receptors; (ii) deletion of the FAR1 gene encoding a proteinthat normally associates with cyclindependent kinases leading to cellcycle arrest; and (iii) construction of reporter genes fused to the FUS1 gene promoter (where FUS 1 encodes a membrane-anchored glycoproteinrequired for cell fusion). Downstream reporter genes can permit either apositive growth selection (e.g., histidine prototrophy using theFUS1-HIS3 reporter), or a calorimetric, fluorimetric orspectrophotometric readout, depending on the specific reporter constructused (e.g., b-galactosidase induction using a FUS1-LacZ reporter).

[0474] The yeast cells can be further engineered to express and secretesmall peptides from random peptide libraries, some of which can permitautocrine activation of heterologously expressed human (or mammalian)G-protein coupled receptors (Broach, J. R. and Thorner, J., Nature 384:14-16, 1996; Manfredi et al., Mol. Cell. Biol. 16: 4700-4709,1996). Thisprovides a rapid direct growth selection (e.g, using the FUS 1-HIS3reporter) for surrogate peptide agonists that activate characterized ororphan receptors. Alternatively, yeast cells that functionally expresshuman (or mammalian) G-protein coupled receptors linked to a reportergene readout (e.g., FUS1-LacZ) can be used as a platform forhigh-throughput screening of known ligands, fractions of biologicalextracts and libraries of chemical compounds for either natural orsurrogate ligands.

[0475] Functional agonists of sufficient potency (whether natural orsurrogate) can be used as screening tools in yeast cell-based assays foridentifying G-protein coupled receptor antagonists. For example,agonists will promote growth of a cell with FUS-HIS3 reporter or givepositive readout for a cell with FUSI-LacZ. However, a candidatecompound which inhibits growth or negates the positive readout inducedby an agonist is an antagonist. For this purpose, the yeast systemoffers advantages over mammalian expression systems due to its ease ofutility and null receptor background (lack of endogenous G-proteincoupled receptors) which often interferes with the ability to identifyagonists or antagonists.

[0476] Therapeutic Assays

[0477] The GPCR proteins according to this invention may play a role incell signaling, in cell cycle regulation, and/or in neurologicaldisorders. The GPCR proteins may further be involved in neoplastic,cardiovascular, and immunological disorders.

[0478] In one embodiment in accordance with the present invention, thenovel GPCR protein may play a role in neoplastic disorders. Anantagonist or inhibitor of the GPCR protein may be administered to anindividual to prevent or treat a neoplastic disorder. Such disorders mayinclude, but are not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, and teratocarcinoma, and particularly,cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast,cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. In arelated aspect, an antibody which specifically binds to GPCR may be useddirectly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress the GPCR polypeptide.

[0479] In yet another embodiment of the present invention, an antagonistor inhibitory agent of the GPCR polypeptide may be administeredtherapeutically to an individual to prevent or treat an immunologicaldisorder. Such disorders may include, but are not limited to, AIDS, HIVinfection, Addison's disease, adult respiratory distress syndrome,allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis,Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjogren's syndrome, and autoimmune thyroiditis;complications of cancer, hemodialysis, extracorporeal circulation;viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections, trauma, and neurological disorders including, but notlimited to, akathesia, Alzheimer's disease, amnesia, amyotrophic lateralsclerosis, bipolar disorder, catatonia, cerebral neoplasms, dementia,depression, Down's syndrome, tardive dyskinesia, dystonias, epilepsy,Huntington's disease, multiple sclerosis, Parkinson's disease, paranoidpsychoses, schizophrenia, and Tourette's disorder.

[0480] A preferred method of treating a GPCR associated disease,disorder, syndrome, or condition in a mammal comprises administration ofa modulator, preferably an inhibitor or antagonist, of a GPCRpolypeptide or homologue of the invention, in an amount effective totreat, reduce, and/or ameliorate the symptoms incurred by theGPCR-associated disease, disorder, syndrome, or condition. In someinstances, an agonist or enhancer of a GPCR polypeptide or homologue ofthe invention is administered in an amount effective to treat and/orameliorate the symptoms incurred by a GPCR-related disease, disorder,syndrome, or condition. In other instances, the administration of anovel GPCR polypeptide or homologue thereof pursuant to the presentinvention is envisioned for administration to treat a GPCR associateddisease.

[0481] In yet another embodiment of the present invention, an expressionvector containing the complement of the polynucleotide encoding a GPCRpolypeptide is administered to an individual to treat or prevent any oneof the types of diseases, disorders, or conditions previously described,in an antisense therapy method.

[0482] The GPCR proteins; modulators, including antagonists, antibodies,and agonists; complementary sequences; or vectors of the presentinvention can also be administered in combination with other appropriatetherapeutic agents as necessary or desired. Selection of the appropriateagents for use in combination therapy may be made by the skilledpractitioner in the art, according to conventional pharmaceutical andclinical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects or adverse events.

[0483] Antagonists or inhibitors of the GPCR polypeptide of thisinvention can be produced using methods which are generally known in theart. In particular, purified GPCR protein, or fragments thereof, can beused to produce antibodies, or to screen libraries of pharmaceuticalagents, to identify those which specifically bind to the novel GPCRpolypeptides as described herein.

[0484] Antibodies specific for GPCR polypeptide, or immunogenic peptidefragments thereof, can be generated using methods that have long beenknown and conventionally practiced in the art. Such antibodies mayinclude, but are not limited to, polyclonal, monoclonal, neutralizingantibodies, (i.e., those which inhibit dimer formation), chimeric,single chain, Fab fragments, and fragments produced by an Fab expressionlibrary. Non-limiting examples of GPCR polypeptides or immunogenicfragments thereof that may be used to generate antibodies are providedin SEQ ID NOs:14-26.

[0485] For the production of antibodies, various hosts, including goats,rabbits, sheep, rats, mice, humans, and others, can be immunized byinjection with one or more of the GPCR polypeptides, or any immunogenicand/or epitope-containing fragment or oligopeptide thereof, which haveimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase the immunological response. Non-limitingexamples of suitable adjuvants include Freund's (incomplete), mineralgels such as aluminum hydroxide or silica, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, KLH, and dinitrophenol. Adjuvants typically used in humansinclude BCG (bacilli Calmette Guérin) and Corynebacterium parvumn.

[0486] Preferably, the GPCR polypeptides, peptides, fragments, oroligopeptides used to induce antibodies to the GPCR polypeptideimmunogens have an amino acid sequence of at least five amino acids inlength, and more preferably, at least 7-10, or more, amino acids. It isalso preferable that the immunogens are identical to a portion of theamino acid sequence of the natural protein; they may also contain theentire amino acid sequence of a small, naturally occurring molecule. Thepeptides, fragments or oligopeptides may comprise a single epitope orantigenic determinant or multiple epitopes. Short stretches of GPCRamino acids may be fused with another protein as carrier, such as KLH,such that antibodies are produced against the chimeric molecule.

[0487] Monoclonal antibodies to the GPCR polypeptides, or immunogenicfragments thereof, may be prepared using any technique which providesfor the production of antibody molecules by continuous cell lines inculture. Such techniques are conventionally used in the art. Theseinclude, but are not limited to, the hybridoma technique, the humanB-cell hybridoma technique, and the EBV-hybridoma technique (G. Kohleret al., 1975, Nature, 256:495-497; D. Kozbor et al., 1985, J. Immunol.Methods, 81:31-42; R. J. Cote et al., 1983, Proc. Natl. Acad. Sci. USA,80:2026-2030; and S. P. Cole et al., 1984, Mol. Cell Biol., 62:109-120).The production of monoclonal antibodies to immunogenic proteins andpeptides is well known and routinely used in the art.

[0488] In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (S. L. Morrison et al., 1984, Proc.Natl. Acad. Sci. USA, 81:6851-6855; M. S. Neuberger et al., 1984,Nature, 312:604-608; and S. Takeda et al., 1985, Nature, 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceGPCR polypeptide-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries (D. R. Burton, 1991, Proc. Natl. Acad. Sci. USA, 88:11120-3).Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (R. Orlandi et al., 1989, Proc. Natl. Acad. Sci. USA,86:3833-3837 and G. Winter et al., 1991, Nature, 349:293-299).

[0489] Antibody fragments, which contain specific binding sites for aGPCR polypeptide, may also be generated. For example, such fragmentsinclude, but are not limited to, F(ab′)₂ fragments which can be producedby pepsin digestion of the antibody molecule and Fab fragments which canbe generated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (e.g., W. D. Huse et al., 1989, Science,254.1275-1281).

[0490] Various immunoassays can be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve measuring the formationof complexes between a GPCR polypeptide and its specific antibody. Atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive with two non-interfering GPCR polypeptide epitopes is suitable,but a competitive binding assay may also be employed (Maddox, supra).

[0491] To induce an immunological response in a mammal, a host animal isinoculated with a GPCR polypeptide, or a fragment thereof, of thisinvention in an amount adequate to produce an antibody and/or a T cellimmune response to protect the animal from a disease or disorderassociated with the expression or production of a GPCR polypeptide. Yetanother aspect of the invention relates to a method of inducingimmunological response in a mammal, if applicable or required. Such amethod comprises delivering GPCR polypeptide via a vector directingexpression of GPCR polynucleotide in vivo in order to induce such animmunological response to produce antibody to protect said animal fromGPCR-related diseases.

[0492] A further aspect of the invention relates to an immunologicalvaccine or immunogen formulation or composition which, when introducedinto a mammalian host, induces an immunological response in that mammalto a GPCR polypeptide wherein the composition comprises a GPCRpolypeptide or GPCR gene. The vaccine or immunogen formulation mayfurther comprise a suitable carrier. Since the GPCR polypeptide may bebroken down in the stomach, it is preferably administered parenterally(including subcutaneous, intramuscular, intravenous, intradermal, etc.,injection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents.

[0493] The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampoules and vials, and may be stored ina freeze-dried condition requiring only the addition of the sterileliquid carrier immediately prior to use. A vaccine formulation may alsoinclude adjuvant systems for enhancing the immunogenicity of theformulation, such as oil-in-water systems and other systems known in theart. The dosage will depend on the specific activity of the vaccine andcan be readily determined by routine experimentation.

[0494] In a specific embodiment, formulations of the present inventionmay further comprise antagonists of P-glycoprotein (also referred to asthe multiresistence protein, or PGP), including antagonists of itsencoding polynucleotides (e.g., antisense oligonucleotides, ribozymes,zinc-finger proteins, etc.). P-glycoprotein is well known for decreasingthe efficacy of various drug administrations due to its ability toexport intracellular levels of absorbed drug to the cell exterior. Whilethis activity has been particularly pronounced in cancer cells inresponse to the administration of chemotherapy regimens, a variety ofother cell types and the administration of other drug classes have beennoted (e.g., T-cells and anti-HIV drugs). In fact, certain mutations inthe PGP gene significantly reduces PGP function, making it less able toforce drugs out of cells. People who have two versions of the mutatedgene—one inherited from each parent—have more than four times less PGPthan those with two normal versions of the gene. People may also haveone normal gene and one mutated one. Certain ethnic populations haveincreased incidence of such PGP mutations. Among individuals from Ghana,Kenya, the Sudan, as well as African Americans, frequency of the normalgene ranged from 73% to 84%. In contrast, the frequency was 34% to 59%among British whites, Portuguese, Southwest Asian, Chinese, Filipino andSaudi populations. As a result, certain ethnic populations may requireincreased administration of PGP antagonist in the formulation of thepresent invention to arrive at the an efficacious dose of thetherapeutic (e.g., those from African descent). Conversely, certainethnic populations, particularly those having increased frequency of themutated PGP (e.g., of Caucasian descent, or non-African descent) mayrequire less pharmaceutical compositions in the formulation due to aneffective increase in efficacy of such compositions as a result of theincreased effective absorption (e.g., less PGP activity) of saidcomposition.

[0495] Moreover, in another specific embodiment, formulations of thepresent invention may further comprise antagonists of OATP2 (alsoreferred to as the multiresistance protein, or MRP2), includingantagonists of its encoding polynucleotides (e.g., antisenseoligonucleotides, ribozymes, zinc-finger proteins, etc.). The inventionalso further comprises any additional antagonists known to inhibitproteins thought to be attributable to a multidrug resistant phenotypein proliferating cells.

[0496] Preferred antagonists that formulations of the present maycomprise include the potent P-glycoprotein inhibitor elacridar, and/orLY-335979. Other P-glycoprotein inhibitors known in the art are alsoencompassed by the present invention.

[0497] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used to increase the efficacy of apharmaceutical composition, either directly or indirectly. Such a usemay be administered in simultaneous conjunction with saidpharmaceutical, or separately through either the same or different routeof administration (e.g., intravenous for the polynucleotide orpolypeptide of the present invention, and orally for the pharmaceutical,among others described herein.).

[0498] In an aspect of the present invention, the polynucleotideencoding a GPCR polypeptide, or any fragment or complement thereof, asdescribed herein may be used for therapeutic purposes. For instance,antisense to a GPCR polynucleotide encoding a GPCR polypeptide, may beused in situations in which it would be desirable to block thetranscription of GPCR mRNA. In particular, cells may be transformed,transfected, or injected with sequences complementary to polynucleotidesencoding GPCR polypeptide. Thus, complementary molecules may be used tomodulate GPCR polynucleotide and polypeptide activity, or to achieveregulation of gene function. Such technology is well known in the art,and sense or antisense oligomers or oligonucleotides, or largerfragments, can be designed from various locations along the coding orcontrol regions of the GPCR polynucleotide sequences encoding the novelGPCR polypeptides.

[0499] Polypeptides used in treatment can also be generated endogenouslyin the subject, in treatment modalities often referred to as “genetherapy”. Thus for example, cells from a subject may be engineered witha polynucleotide, such as DNA or RNA, to encode a polypeptide ex vivo,for example, by the use of a retroviral plasmid vector. The cells canthen be introduced into the subject's body in which the desiredpolypeptide is expressed.

[0500] A gene encoding a GPCR polypeptide can be turned off bytransforming a cell or tissue with an expression vector that expresseshigh levels of a GPCR polypeptide-encoding polynucleotide, or a fragmentthereof. Such constructs may be used to introduce untranslatable senseor antisense sequences into a cell. Even in the absence of integrationinto the DNA, such vectors may continue to transcribe RNA moleculesuntil they are disabled by endogenous nucleases. Transient expressionmay last for a month or more with a non-replicating vector, and evenlonger if appropriate replication elements are designed to be part ofthe vector system.

[0501] Modifications of gene expression can be obtained by designingantisense molecules or complementary nucleic acid sequences (DNA, RNA,or PNA), to the control, 5′, or regulatory regions of a GPCRpolynucleotide sequence encoding a GPCR polypeptide, (e.g., a signalsequence, promoters, enhancers, and introns). Oligonucleotides may bederived from the transcription initiation site, for example, betweenpositions −10 and +10 from the start site.

[0502] Similarly, inhibition can be achieved using “triple helix”base-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described (see, for example, J. E. Gee et al., 1994, In: B. E.Huber and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecule orcomplementary sequence may also be designed to block translation of mRNAby preventing the transcript from binding to ribosomes.

[0503] Many methods for introducing vectors into cells or tissues areavailable and are equally suitable for use in vivo, in vitro, and exvivo. For ex vivo therapy, vectors may be introduced into stem cells orbone marrow cells obtained from the patient and clonally propagated forautologous transplant back into that same patient. Delivery bytransfection, direct injection (e.g., microparticle bombardment) and byliposome injections may be achieved using methods which are well knownin the art.

[0504] Any of the therapeutic methods described above can be applied toany individual in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0505] Administration

[0506] A further embodiment of the present invention embraces theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, diluent, or excipient, to achieveany of the above-described therapeutic uses and effects. Suchpharmaceutical compositions can comprise GPCR nucleic acid, polypeptide,or peptides, antibodies to GPCR polypeptide, mimetics, GPCR modulators,such as agonists, antagonists, or inhibitors of a GPCR polypeptide orpolynucleotide. The compositions can be administered alone, or incombination with at least one other agent or reagent, such as astabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs, hormones, or biological response modifiers.

[0507] The pharmaceutical compositions for use in the present inventioncan be administered by any number of routes including, but not limitedto, oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, orrectal means.

[0508] In addition to the active ingredients (e.g., GPCR nucleic acid orpolypeptide, or functional fragments thereof, or a GPCR agonist orantagonist), the pharmaceutical compositions may containpharmaceutically acceptable/physiologically suitable carriers orexcipients comprising auxiliaries which facilitate processing of theactive compounds into preparations that can be used pharmaceutically.Further details on techniques for formulation and administration areprovided in the latest edition of Remington's Pharmaceutical Sciences(Mack Publishing Co., Easton, Pa.).

[0509] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0510] In addition, pharmaceutical preparations for oral use can beobtained by the combination of active compounds with a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores. Suitable excipients are carbohydrate or proteinfillers, such as sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose, such as methyl cellulose, hydroxypropyl-methylcellulose, orsodium carboxymethylcellulose; gums, including arabic and tragacanth,and proteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or a physiologically acceptable saltthereof, such as sodium alginate.

[0511] Dragee cores may be used in conjunction with physiologicallysuitable coatings, such as concentrated sugar solutions, which may alsocontain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for product identification,or to characterize the quantity of active compound, i.e., dosage.

[0512] Pharmaceutical preparations, which can be used orally, furtherinclude push-fit capsules made of gelatin, as well as soft, scaledcapsules made of gelatin and a coating, such as glycerol or sorbitol.Push-fit capsules can contain active ingredients mixed with fillers orbinders, such as lactose or starches, lubricants, such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0513] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances, which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. In addition, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyloleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0514] For topical or nasal administration, penetrants or permeationagents (enhancers) that are appropriate to the particular barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

[0515] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0516] A pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Saltstend to be more soluble in aqueous solvents, or other protonic solvents,than are the corresponding free base forms. In other cases, thepreferred preparation may be a lyophilized powder which may contain anyor all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7%mannitol, at a pH range of 4.5 to 5.5, combined with a buffer prior touse. After the pharmaceutical compositions have been prepared, they canbe placed in an appropriate container and labeled for treatment of anindicated condition. For administration of a GPCR product, such labelingwould include amount, frequency, and method of administration.

[0517] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve the intended purpose. Thedetermination of an effective dose or amount is well within thecapability of those skilled in the art. For any compound, thetherapeutically effective dose can be estimated initially either in cellculture assays, for example, using neoplastic cells, or in animalmodels, usually mice, rabbits, dogs, or pigs. The animal model may alsobe used to determine the appropriate concentration range and route ofadministration. Such information can then be used and extrapolated todetermine useful doses and routes for administration in humans.

[0518] A therapeutically effective dose refers to that amount of activeingredient, for example, GPCR polynucleotide, GPCR polypeptide, orfragments thereof, antibodies to GPCR polypeptide, agonists, antagonistsor inhibitors of GPCR polypeptide, which ameliorates, reduces,diminishes, or eliminates the symptoms or condition. Therapeuticefficacy and toxicity can be determined by standard pharmaceuticalprocedures in cell cultures or in experimental animals, e.g., ED₅₀ (thedose therapeutically effective in 50% of the population) and LD₅₀ (thedose lethal to 50% of the population). The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe ratio, LD₅₀/ED₅₀. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used in determining a range of dosages forhuman use. Preferred dosage contained in a pharmaceutical composition iswithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

[0519] The practitioner, who will consider the factors related to anindividual requiring treatment, will determine the exact dosage. Dosageand administration are adjusted to provide sufficient levels of theactive component, or to maintain the desired effect. Factors which maybe taken into account include the severity of the individual's diseasestate; the general health of the patient; the age, weight, and gender ofthe patient; diet; time and frequency of administration; drugcombination(s); reaction sensitivities; and tolerance/response totherapy. As a general guide, long-acting pharmaceutical compositions maybe administered every 3 to 4 days, every week, or once every two weeks,depending on half-life and clearance rate of the particular formulation.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, as is well understood in the art.

[0520] As a guide, normal dosage amounts may vary from 0.1 to 100,000micrograms (μg), up to a total dose of about 1 gram (g), depending uponthe route of administration. Guidance as to particular dosages andmethods of delivery is provided in the literature and is generallyavailable to practitioners in the art. Those skilled in the art willemploy different formulations for nucleotides than for proteins or theirinhibitors or activators. Similarly, the delivery of polynucleotides orpolypeptides will be specific to particular cells, conditions,locations, and the like.

[0521] Microarrays and Screening Assays

[0522] In another embodiment of the present invention, oligonucleotides,or longer fragments derived from the GPCR polynucleotide sequencesdescribed herein can be used as targets in a microarray. The microarraycan be used to monitor the expression levels of large numbers of genessimultaneously (to produce a transcript image), and to identify geneticvariants, mutations and polymorphisms. This information may be used todetermine gene function, to understand the genetic basis of a disease,to diagnose disease, and to develop and monitor the activities oftherapeutic agents. In a particular aspect, the microarray is preparedand used according to the methods described in WO 95/11995 (Chee etal.); D. J. Lockhart et al., 1996, Nature Biotechnology, 14:1675-1680;and M. Schena et al., 1996, Proc. Natl. Acad. Sci. USA, 93:10614-10619).Microarrays are further described in U.S. Pat. No. 6,015,702 to P. Lalet al.

[0523] In another embodiment of this invention, a nucleic acid sequencewhich encodes a novel GPCR polypeptide, may also be used to generatehybridization probes, which are useful for mapping the naturallyoccurring genomic sequence. The sequences may be mapped to a particularchromosome, to a specific region of a chromosome, or to artificialchromosome constructions (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial PI constructions, orsingle chromosome cDNA libraries, as reviewed by C. M. Price, 1993,Blood Rev., 7:127-134 and by B. J. Trask, 1991, Trends Genet.,7:149-154.

[0524] In another embodiment of the present invention, a GPCRpolypeptide of this invention, its catalytic or immunogenic fragments,or oligopeptides thereof, can be used for screening libraries ofcompounds in any of a variety of drug screening techniques. The fragmentemployed in such screening may be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. Theformation of binding complexes, between the GPCR polypeptide, or aportion thereof, and the agent being tested, may be measured utilizingtechniques commonly practiced in the art.

[0525] Another technique for drug screening, which may be employed,provides for high throughput screening of compounds having suitablebinding affinity to the protein of interest as described in WO 84/03564(Venton, et al.). In this method, as applied to the GPCR protein, largenumbers of different small test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with the GPCR polypeptide, or fragments thereof,and washed. Bound GPCR polypeptide is then detected by methods wellknown in the art. Purified GPCR polypeptide can also be coated directlyonto plates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

[0526] In a further embodiment, competitive drug screening assays can beused in which neutralizing antibodies, capable of binding a GPCRpolypeptide according to this invention, specifically compete with atest compound for binding to the GPCR polypeptide. In this manner, theantibodies can be used to detect the presence of any peptide that sharesone or more antigenic determinants with the GPCR polypeptide.

[0527] The human Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13polypeptides and/or peptides of the present invention, or immunogenicfragments or oligopeptides thereof, can be used for screeningtherapeutic drugs or compounds in a variety of drug screeningtechniques. The fragment employed in such a screening assay may be freein solution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The reduction or abolition of activity of theformation of binding complexes between the ion channel protein and theagent being tested can be measured. Thus, the present invention providesa method for screening or assessing a plurality of compounds for theirspecific binding affinity with a Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or 13 polypeptide, or a bindable peptide fragment, of thisinvention, comprising providing a plurality of compounds, combining theGene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 polypeptide, or abindable peptide fragment, with each of a plurality of compounds for atime sufficient to allow binding under suitable conditions and detectingbinding of the Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13polypeptide or peptide to each of the plurality of test compounds,thereby identifying the compounds that specifically bind to the Gene 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 polypeptide or peptide.

[0528] Methods of identifying compounds that modulate the activity ofthe novel human Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13polypeptides and/or peptides are provided by the present invention andcomprise combining a potential or candidate compound or drug modulatorof GPCR biological activity with an Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or 13 polypeptide or peptide, for example, the Gene 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid sequence as set forth in SEQID NO:2, and measuring an effect of the candidate compound or drugmodulator on the biological activity of the Gene 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, or 13 polypeptide or peptide.

[0529] Such measurable effects include, for example, physical bindinginteraction; the ability to cleave a suitable GPCR substrate; effects onnative and cloned Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or13-expressing cell line; and effects of modulators or otherGPCR-mediated physiological measures.

[0530] Another method of identifying compounds that modulate thebiological activity of the novel Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or 13 polypeptides of the present invention comprises combining apotential or candidate compound or drug modulator of a GPCR biologicalactivity with a host cell that expresses the Gene 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, or 13 polypeptide and measuring an effect of thecandidate compound or drug modulator on the biological activity of theGene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 polypeptide. The hostcell can also be capable of being induced to express the Gene 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 polypeptide, e.g., via inducibleexpression. Physiological effects of a given modulator candidate on theGene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 polypeptide can alsobe measured. Thus, cellular assays for particular GPCR modulators may beeither direct measurement or quantification of the physical biologicalactivity of the Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13polypeptide, or they may be measurement or quantification of aphysiological effect. Such methods preferably employ a Gene 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, or 13 polypeptide as described herein, or anoverexpressed recombinant Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or13 polypeptide in suitable host cells containing an expression vector asdescribed herein, wherein the Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or 13 polypeptide is expressed, overexpressed, or undergoesupregulated expression.

[0531] Another aspect of the present invention embraces a method ofscreening for a compound that is capable of modulating the biologicalactivity of a Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13polypeptide, comprising providing a host cell containing an expressionvector harboring a nucleic acid sequence encoding a Gene 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, or 13 polypeptide, or a functional peptide orportion thereof (e.g., SEQ ID NOS:2); determining the biologicalactivity of the expressed Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or13 polypeptide in the absence of a modulator compound; contacting thecell with the modulator compound and determining the biological activityof the expressed Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13polypeptide in the presence of the modulator compound. In such a method,a difference between the activity of the Gene 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, or 13 polypeptide in the presence of the modulator compoundand in the absence of the modulator compound indicates a modulatingeffect of the compound.

[0532] Essentially any chemical compound can be employed as a potentialmodulator or ligand in the assays according to the present invention.Compounds tested as GPCR modulators can be any small chemical compound,or biological entity (e.g., protein, sugar, nucleic acid, lipid). Testcompounds will typically be small chemical molecules and peptides.Generally, the compounds used as potential modulators can be dissolvedin aqueous or organic (e.g., DMSO-based) solutions. The assays aredesigned to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source. Assays aretypically run in parallel, for example, in microtiter formats onmicrotiter plates in robotic assays. There are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs, Switzerland), for example. Also, compounds may besynthesized by methods known in the art.

[0533] High throughput screening methodologies are particularlyenvisioned for the detection of modulators of the novel Gene 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, or 13 polynucleotides and polypeptidesdescribed herein. Such high throughput screening methods typicallyinvolve providing a combinatorial chemical or peptide library containinga large number of potential therapeutic compounds (e.g., ligand ormodulator compounds). Such combinatorial chemical libraries or ligandlibraries are then screened in one or more assays to identify thoselibrary members (e.g., particular chemical species or subclasses) thatdisplay a desired characteristic activity. The compounds so identifiedcan serve as conventional lead compounds, or can themselves be used aspotential or actual therapeutics.

[0534] A combinatorial chemical library is a collection of diversechemical compounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (i.e.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library, is formed by combininga set of chemical building blocks in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

[0535] The preparation and screening of combinatorial chemical librariesis well known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g. U.S. Pat.No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; andHoughton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptoids(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagiharaet al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J.Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of smallcompound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661),oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidylphosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) andPCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996,Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

[0536] Devices for the preparation of combinatorial libraries arecommercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A AppliedBiosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.).In addition, a large number of combinatorial libraries are commerciallyavailable (e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, Russia;Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., and thelike).

[0537] In one embodiment, the invention provides solid phase based invitro assays in a high throughput format, where the cell or tissueexpressing an ion channel is attached to a solid phase substrate. Insuch high throughput assays, it is possible to screen up to severalthousand different modulators or ligands in a single day. In particular,each well of a microtiter plate can be used to perform a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 96 modulators. If 1536 well plates are used, then a single platecan easily assay from about 100 to about 1500 different compounds. It ispossible to assay several different plates per day; thus, for example,assay screens for up to about 6,000-20,000 different compounds arepossible using the described integrated systems.

[0538] In another of its aspects, the present invention encompassesscreening and small molecule (e.g., drug) detection assays which involvethe detection or identification of small molecules that can bind to agiven protein, i.e., a Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13polypeptide or peptide. Particularly preferred are assays suitable forhigh throughput screening methodologies.

[0539] In such binding-based detection, identification, or screeningassays, a functional assay is not typically required. All that is neededis a target protein, preferably substantially purified, and a library orpanel of compounds (e.g., ligands, drugs, small molecules) or biologicalentities to be screened or assayed for binding to the protein target.Preferably, most small molecules that bind to the target protein willmodulate activity in some manner, due to preferential, higher affinitybinding to functional areas or sites on the protein.

[0540] An example of such an assay is the fluorescence based thermalshift assay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano etal.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assayallows the detection of small molecules (e.g., drugs, ligands) that bindto expressed, and preferably purified, ion channel polypeptide based onaffinity of binding determinations by analyzing thermal unfolding curvesof protein-drug or ligand complexes. The drugs or binding moleculesdetermined by this technique can be further assayed, if desired, bymethods, such as those described herein, to determine if the moleculesaffect or modulate function or activity of the target protein.

[0541] To purify a Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13polypeptide or peptide to measure a biological binding or ligand bindingactivity, the source may be a whole cell lysate that can be prepared bysuccessive freeze-thaw cycles (e.g., one to three) in the presence ofstandard protease inhibitors. The Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or 13 polypeptide may be partially or completely purified bystandard protein purification methods, e.g., affinity chromatographyusing specific antibody described infra, or by ligands specific for anepitope tag engineered into the recombinant Gene 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, or 13 polypeptide molecule, also as described herein.Binding activity can then be measured as described.

[0542] Compounds which are identified according to the methods providedherein, and which modulate or regulate the biological activity orphysiology of the Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13polypeptides according to the present invention are a preferredembodiment of this invention. It is contemplated that such modulatorycompounds may be employed in treatment and therapeutic methods fortreating a condition that is mediated by the novel Gene 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, or 13 polypeptides by administering to anindividual in need of such treatment a therapeutically effective amountof the compound identified by the methods described herein.

[0543] In addition, the present invention provides methods for treatingan individual in need of such treatment for a disease, disorder, orcondition that is mediated by the Gene 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or 13 polypeptides of the invention, comprising administering tothe individual a therapeutically effective amount of the Gene 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, or 13-modulating compound identified by amethod provided herein.

EXAMPLES

[0544] The Examples herein are meant to exemplify the various aspects ofcarrying out the invention and are not intended to limit the scope ofthe invention in any way.

[0545] The Examples do not include detailed descriptions forconventional methods employed, such as in the construction of vectors,the insertion of cDNA into such vectors, or the introduction of theresulting vectors into the appropriate host. Such methods are well knownto those skilled in the art and are described in numerous publications,for example, Sambrook, Fritsch, and Maniatis, Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, Cold Spring Harbor Laboratory Press,USA, (1989).

Example 1 Bioinformatics Analysis

[0546] Currently, one approach used for identifying and characterizingthe genes distributed along the human genome includes utilizing largefragments of genomic DNA which are isolated, cloned, and sequenced.Potential open reading frames in these genomic sequences were identifiedusing bioinformatics software.

[0547] GPCR sequences were obtained from the GPCR database at EuropeanMolecular Biology Laboratory (EMBL) (http://www.7tm.org/gpcr/). Thesesequences (more than 1300 protein sequences) were used as probes tosearch the human genomic, public and private EST databases. The searchprogram used was BLAST2. The alignment was performed using the BLAST 2algorithm according to the default parameters (S. F. Altschul, et al.,Nucleic Acids Res. 25:3389-3402, 1997). The BLAST results were analyzedfor potential novel GPCR candidates. The candidate sequences, fromgenomic or EST data, were then characterized. The characterizationmethods include sequence and profile-based analyses. The functionalprediction is based on sequence identity and homology and/or domaininformation. The “query” sequence represents the novel GPCR amino acidsequence of the invention; the subject (“sbjct”) sequence represents thelocal matching sequence of the protein found in the database. The aminoacids between the query and target sequences represent matchingidentical amino acids between the two sequences. Plus signs (“+”)between the query and target sequences represent similar amino acidsbetween the two sequences. Spaces between the query and the targetsequences indicate regions of non-identity for the aligned polypeptides.

[0548]FIG. 27A shows the regions of local identity (50%) and similarity(66%) between the novel human GPCR Gene 1 encoded amino acid sequence(SEQ ID NO:14, FIG. 2) of the present invention and the human olfactoryreceptor 5U1, i.e., the “sbjt” sequence (SEQ ID NO:72). For Gene 1, adomain prediction was also determined (FIG. 27B). The results suggestthat the Gene 1 GPCR polypeptide of this invention represents a novelmember of the rhodopsin protein family. Based upon this prediction, itis expected that the Gene 1 GPCR polypeptide may share at least somebiological activity with members of the rhodopsin family oftransmembrane receptors, in addition to specific members known in theart, or as otherwise described herein.

[0549]FIG. 28A shows the regions of local identity (98%) and similarity(98%) between the novel human GPCR Gene 2 encoded amino acid sequence(SEQ ID NO:15, FIG. 4) of the present invention and the human Gprotein-coupled receptor 61, i.e., the “sbjt” sequence (SEQ ID NO:73).FIG. 28B shows the regions of local identity (98%) and similarity (98%)between the novel human GPCR Gene 2 encoded amino acid sequence (SEQ IDNO:15, FIG. 4) of the present invention and a portion of rabbit Gprotein-coupled receptor protein, i.e., the “sbjt” sequence (SEQ IDNO:74). For Gene 2, a domain prediction was also determined (FIG. 28C).The results suggest that the Gene 2 GPCR polypeptide of this inventionrepresents a novel member of the rhodopsin protein family. Based uponthis prediction, it is expected that the Gene 2 GPCR polypeptide mayshare at least some biological activity with members of the rhodopsinfamily of transmembrane receptors, in addition to specific members knownin the art, or as otherwise described herein.

[0550]FIG. 29A shows the regions of local identity (48%) and similarity(61%) between the novel human GPCR Gene 3 encoded amino acid sequence(SEQ ID NO:16, FIG. 6) of the present invention and mouse olfactoryreceptor protein 3′Beta4, i.e., the “sbjt” sequence (SEQ ID NO:75). ForGene 3, a domain prediction was also determined (FIG. 29B). The resultssuggest that the Gene 3 GPCR polypeptide of this invention represents anovel member of the rhodopsin protein family. Based upon thisprediction, it is expected that the Gene 3 GPCR polypeptide may share atleast some biological activity with members of the rhodopsin family oftransmembrane receptors, in addition to specific members known in theart, or as otherwise described herein.

[0551]FIG. 30A shows the regions of local identity (48%) and similarity(64%) between the novel human GPCR Gene 4 encoded amino acid sequence(SEQ ID NO:17, FIG. 8) of the present invention and mouse olfactoryreceptor protein 3′Beta1, i.e., the “sbjt” sequence (SEQ ID NO:76). ForGene 4, a domain prediction was also determined (FIG. 30B). The resultssuggest that the Gene 4 GPCR polypeptide of this invention represents anovel member of the rhodopsin protein family. Based upon thisprediction, it is expected that the Gene 4 GPCR polypeptide may share atleast some biological activity with members of the rhodopsin family oftransmembrane receptors, in addition to specific members known in theart, or as otherwise described herein.

[0552]FIG. 31 shows the regions of local identity (44%) and similarity(61%) between the novel human GPCR Gene 5 encoded amino acid sequence(SEQ ID NO: 18, FIG. 10) of the present invention and human tastereceptor T2R13, i.e., the “sbjt” sequence (SEQ ID NO:77). FIG. 32 showsthe regions of local identity (26%) and similarity (47%) between thenovel human GPCR Gene 6 encoded amino acid sequence (SEQ ID NO:19, FIG.12) of the present invention and human chemokine receptor 1, i.e., the“sbjt” sequence (SEQ ID NO:78). FIG. 33 shows the regions of localidentity (77%) and similarity (82%) between the novel human GPCR Gene 7encoded amino acid sequence (SEQ ID NO:20, FIG. 14) of the presentinvention and human G protein-coupled receptor hHI7T213, i.e., the“sbjt” sequence (SEQ ID NO:79). FIG. 34 shows the regions of localidentity (32%) and similarity (54%) between the novel human GPCR Gene 8encoded amino acid sequence (SEQ ID NO:21, FIG. 16) of the presentinvention and human G protein-coupled receptor RE2, i.e., the “sbjt”sequence (SEQ ID NO:80). FIG. 35 shows the regions of local identity(47%) and similarity (67%) between the novel human GPCR Gene 9 encodedamino acid sequence (SEQ ID NO:22, FIG. 18) of the present invention andolfactory receptor OR93Gib, i.e., the “sbjt” sequence (SEQ ID NO:81).FIG. 36 shows the regions of local identity (76%) and similarity (87%)between the novel human GPCR Gene 10 encoded amino acid sequence (SEQ IDNO:23, FIG. 20) of the present invention and mouse odorant receptor K11,i.e., the “sbjt” sequence (SEQ ID NO:82). FIG. 37 shows the regions oflocal identity (78%) and similarity (85%) between the novel human GPCRGene 11 encoded amino acid sequence (SEQ ID NO:24, FIG. 22) of thepresent invention and mouse odorant receptor K4h11, i.e., the “sbjt”sequence (SEQ ID NO:83). FIG. 38 shows the regions of local identity(43%) and similarity (59%) between the novel human GPCR Gene 12 encodedamino acid sequence (SEQ ID NO:25, FIG. 24) of the present invention andmouse vomeronasal receptor V1RC3, i.e., the “sbjt” sequence (SEQ IDNO:84). FIG. 39 shows the regions of local identity (30%) and similarity(53%) between the novel human GPCR Gene 13 encoded amino acid sequence(SEQ ID NO:26, FIG. 26) of the present invention and human very large Gprotein-coupled receptor-1, i.e., the “sbjt” sequence (SEQ ID NO:85).

[0553] In the case of Gene 13, the top genomic exon hits from the BLASTresults were searched back against the non-redundant protein and patentsequence databases. From this analysis, exons encoding novel GPCR's wereidentified based on sequence homology. Also, the genomic regionsurrounding the matching exons was analyzed. Based on this analysis, thepotential full length nucleotide sequence of Gene 13 (SEQ ID NO:13,FIGS. 25A-B) of the novel human GPCR, Gene 13, also referred to asGPCR-P20 and GPCR-P151, was identified directly from the genomicsequence (Genbank Acc ID:AC068323).

[0554] The amino acid sequence of the Gene 13 polypeptide (SEQ ID NO:26)encoded by the Gene 13 polynucleotide sequence (SEQ ID NO:13) wassearched against the non-redundant protein and patent sequencedatabases. The alignment of Gene 13 polypeptide sequence (SEQ ID NO:26)with the top matching hits was performed using the GCG pileup program.The GAP global alignment program in GCG was used to calculate thepercent identity and similarity values. The GAP program uses analgorithm based on (S. B. Needleman, C. D. Wunsch, J. Mol. Biol.48(3):443-53, 1970), and the following parameters in the program wasused: gap creation penalty:6 and gap extension penalty:2. In thealignment results, the blackened areas represent identical amino acids,the grey highlighted areas represent similar amino acids and dottedareas represent gaps in more than half of the listed sequences.

[0555] FIGS. 40A-40E show the regions of local identity (29.7%) andsimilarity (41.6%) between the novel human Gene 13 encoded amino acidsequence (SEQ ID NO:26, FIGS. 25A-B) of the present invention and thehuman GPCR receptor human_hypothetical 1, (Genbank Acc ID: 12044471; SEQID NO:149). Also shown in FIGS. 40A-40E are the regions of localidentity (29.7%) and similarity (41.6%) between the novel human Gene 13encoded amino acid sequence (SEQ ID NO:26, FIGS. 25A-B) of the presentinvention and the human GPCR receptor, human_hypothetical 2 (Genbank AccID: 14729798; SEQ ID NO:150)). These results indicate that the Gene 13polypeptide of this invention represents a novel member of the GPCRprotein family. It is thus expected that the Gene 13 polypeptide sharesat least some biological activity with members of the GPCR family oftransmembrane receptors, in addition to specific members known in theart, or as otherwise described herein.

[0556] The sequence information from the novel gene candidates was usedfor full-length cloning and expression profiling. Primer sequences wereobtained using the primer3 program (Steve Rozen, Helen J. Skaletsky(1996, 1997) Primer3. Code available athttp://www-genome.wi.mit.edu/genome_software/other/primer3.html). Theright and left primers as presented in Table 2 were used in the cloningprocess and the “internal oligo” as presented in Table 3 was used as ahybridization probe to detect the PCR product after amplification.

Example 2 Cloning of the Novel Human GPCRs

[0557] Using the EST sequence, an antisense oligonucleotide with biotinon the 5′ end complementary to the putative coding region of GPCR wasdesigned. This biotinylated oligo can be incubated with a mixture ofsingle-stranded covalently closed circular cDNA libraries, which containDNA corresponding to the sense strand. Hybrids between the biotinylatedoligo and the circular cDNA are captured on streptavidin magnetic beads.Upon thermal release of the cDNA from the biotinylated oligo, the singlestranded cDNA is converted into double strands using a primer homologousto a sequence on the cDNA cloning vector. The double stranded cDNA isintroduced into E. coli by electroporation and the resulting coloniesare screened by PCR, using a primer pair designed from the EST sequenceto identify the proper cDNA. Oligos used to identify the cDNA of a GPCRgene of this invention by PCR can be selected from any one of GPCRsequences as represented in SEQ ID NOs:1-13.

Example 3 Multiplex Cloning

[0558] Construction of Size Fractionated cDNA Libraries

[0559] PolyA+ RNA was purchased from Clontech, treated with DNase I toremove traces of genomic DNA contamination and converted into doublestranded cDNA using the SuperScript™ Plasmid System for cDNA Synthesisand Plasmid Cloning (Life Technologies). No radioisotope wasincorporated in either of the cDNA synthesis steps. The cDNA was thensize fractionated on a TransGenomics HPLC system equipped with a sizeexclusion column (TosoHass) with dimensions of 7.8 mm×30 cm and aparticle size of 10 μm. Tris buffered saline (TBS) was used as themobile phase, and the column was run at a flow rate of 0.5 mL/min. Thesystem was calibrated by running a 1 kb ladder through the column andanalyzing the fractions by agarose gel electrophoresis. Using thesedata, it can be determine which fractions are to be pooled to obtain thelargest cDNA library. Generally, fractions that eluted in the range of12 to 15 minutes were used.

[0560] The cDNA was precipitated, concentrated and then ligated into theSalI/NotI sites in pSPORT. After electroporation into E. coli DH12S,colonies were subjected to a miniprep procedure and the resulting cDNAwas digested using SalI/NotI restriction enzymes. Generally, the averageinsert size of libraries made in this fashion was greater the 3.5 Kb;the overall complexity of the library is optimally greater than10⁷.independent clones. The library was amplified in semi-solid agar for2 days at 30° C. An aliquot (200 microliters) of the amplified librarywas inoculated into a 200 mL culture for single-stranded DNA isolationby super-infection with an f1 helper phage. The single stranded circularDNA was concentrated by ethanol precipitation, resuspended at aconcentration of one microgram per microliter and used for the cDNAcapture experiments.

[0561] Conversion of Double-Stranded cDNA Libraries into Single-StrandedCircular Form

[0562] To prepare cultures, 200 mL LB with 400 μL carbenicillin (100mg/mL stock solution) was inoculated with from 200 μL to 1 mL of thawedcDNA library and incubated at 37° C. while shaking at 250 rpm forapproximately 45 minutes, or until an OD600 of 0.025-0.040 was attained.M13K07 helper phage (1 mL) was added to the culture and grown for 2hours, after which Kanamycin (500 μl; 30 mg/mL) was added and theculture was grown for an additional 15-18 hours.

[0563] The culture was then poured into 6 screw-cap tubes (50 mLautoclaved tubes) and cells subjected to centrifugation at 10K in anHB-6 rotor for 15 minutes at 4° C. to pellet the cells. The supernatantwas filtered through a 0.2 μm filter and 12,000 units of Gibco DNase Iwas added. The mixture was incubated for 90 minutes at room temperature.

[0564] For PEG precipitation, 50 mL of ice-cold 40% PEG 8000, 2.5 MNaCl, and 10 mM MgSO₄ were added to the supernatant, mixed, andaliquotted into 6 centrifuge tubes (covered with parafilm). The tubesand contents were incubated for 1 hour on wet ice or at 4° C. overnight.The tubes were then centrifuged at 10K in a HB-6 rotor for 20 minutes at4° C. to pellet the helper phage.

[0565] Following centrifugation, the supernatant was discarded and thesides of the tubes were dried. Each pellet was resuspended in 1 mL TE,pH 8. The resuspended pellets were pooled into a 14 mL tube (Sarstadt),(6 mL total). SDS was added to 0.1% (60 μl of stock 10% SDS). Freshlymade proteinase K (20 mg/mL) was added (60 μl) and the suspension wasincubated for 1 hour at 42° C.

[0566] For phenol/chloroform extractions, 1 mL of NaCl (5M) was added tothe suspension in the tube. An equal volume of phenol/chloroform (6 mL)was added and the contents were vortexed or shaken. The suspension wasthen centrifuged at 5K in an HB-6 rotor for 5 minutes at 4° C. Theaqueous (top) phase was transferred to a new tube (Sarstadt) andextractions were repeated until no interface was visible.

[0567] Ethanol precipitation was then performed on the aqueous phasewhose volume was divided into 2 tubes (3 mL each). To each tube, 2volumes of 100% ethanol was added and precipitation was carried outovernight at −20° C. The precipitated DNA was pelleted at 10K in an HB-6rotor for 20 minutes at 4° C. The ethanol was discarded. Each pellet wasresuspended in 700 μl of 70% ethanol. The contents of each tube werecombined into one micro centrifuge tube and centrifuged in a microcentrifuge (Eppendorf) at 14K for 10 minutes at 4° C. After discardingthe ethanol, the DNA pellet was dried in a speed vacuum. In order toremove oligosaccharides, the pellet was resuspended in 50 μl TE buffer,pH 8. The resuspension was incubated on dry ice for 10 minutes andcentrifuged at 14K in an Eppendorf microfuge for 15 minutes at 4° C. Thesupernatant was then transferred to a new tube and the final volume wasrecorded.

[0568] To check purity, DNA was diluted 1:100 and added to a microquartz cuvette, where DNA was analyzed by spectrometry at anOD260/OD280. The preferred purity ratio was between 1.7 and 2.0. The DNAwas diluted to 1 μg/μL in TE, pH 8 and stored at 4° C. The concentrationof DNA was calculated using the formula: (32 μg/mL*OD)(mL/1000μL)(100)(OD260). The quality of single-stranded DNA was determined byfirst mixing 1 μL of 5 ng/μl ssDNA; 11 μL deionized water; 1.5 μL 10 μMT7 sport primer (fresh dilution of stock); 1.5 μl 10×Precision-Taqbuffer per reaction. In the repair mix, a cocktail of 4 μl of 5 mM dNTPs(1.25 mM each); 1.5 μL 10×Precision-Taq buffer; 9.25μL deionized water;and 0.25 μL Precision-Taq polymerase was mixed per reaction andpreheated at 70° C. until the middle of the thermal cycle.

[0569] The DNA mixes were aliquotted into PCR tubes and the thermalcycle was started. The PCR thermal cycle consisted of 1 cycle at 95° C.for 20 sec.; 59° C. for 1 min. (15 μL repair mix added); and 73° C. for23 minutes. For ethanol precipitation, 15 μg glycogen, 16 μl ammoniumacetate (7.5M), and 125 μL 100% ethanol were added and the contents werecentrifuged at 14K in an Eppendorf microfuge for 30 minutes at 4° C. Theresulting pellet was washed 1 time with 125 μL 70% ethanol and then theethanol was discarded. The pellet was dried in a speed vacuum andresuspended in 10 μL TE buffer, pH 8.

[0570] Single-stranded DNA was electroporated into E. coli DH10B orDH12S cells by pre-chilling the cuvettes and sliding holder and thawingthe cells on ice-water. DNA was aliquotted into micro centrifuge tubes(Eppendorf) as follows: 2 μL repaired library, (=1×10⁻³ μg); 1 μLunrepaired library (1 ng/μL), (=1×10⁻³ μg); and 1 μL pUC19 positivecontrol DNA (0.01 μg/μL), (=1×10⁻⁵ μg). The mixtures were stored on iceuntil use.

[0571] One at a time, 40 μL of cells were added to a DNA aliquot. Thecell/DNA mixture was not pipetted up and down more than one time. Themixture was then transferred via pipette into a cuvette between themetal plates and electroporation was performed at 1.8 kV. Immediatelyafterward, 1 mL SOC medium (i.e., SOB (bacto-tryptone; bacto-yeastextract; NaCl)+glucose (20 mM)+Mg²⁺) (See, J. Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., A.2, 1989) was added to thecuvette and the contents were transferred 15 mL media as commonly knownin the art. The cells were allowed to recover for 1 hour at 37° C. withshaking (225 rpm).

[0572] Serial dilutions of the culture were made in 1:10 increments (20μL into 180 μL LB) for plating the electroporated cells. For therepaired library, dilutions of 1:100, 1:1000, 1:10,000 were made. Forthe unrepaired library, dilutions of 1:10 and 1:100 were made. Positivecontrol dilutions of 1:10 and 1:100 were made. Each dilution (100 μL)was plated onto small plates containing LB+carbenicillin and incubatedat 37° C. overnight. The titer and background were calculated by methodswell known in the art. Specifically, the colonies on each plate werecounted using the lowest dilution countable. The titer was calculatedusing the formula: (# of colonies)(dilution factor)(200 μL/100 μL)(1000μL/20 μL)=CFUs, where CFUs/μg DNA used=CFU/μg. The %background=((unrepaired CFU/μg)/(repaired CFU/μg))×100%.

[0573] Solution Hybridization and DNA Capture

[0574] One microliter of anti-sense biotinylated oligonucleotides (orsense oligonucleotides when annealing to single-stranded DNA frompSPORT2 vector) containing 150 ng of up to 50 different 80-meroligonucleotide probes was added to 6 μL (6 μg) of a mixture of up to 15single-stranded, covalently-closed, circular cDNA libraries and 7 μL of100% formamide in a 0.5 mL PCR tube.

[0575] In the case of Gene 13, the following 80′mer oligonucleotideswere used to clone the full-length Gene 13 polynucleotide:TCATGGAACTCTGTCTCCAGTGACTTTGCATTGG (SEQ ID NO:155)AACATAGACTCTGATCCTGATGGTGATCTCGCCT TCACCTCTGGCA-3′ and5′-TTGGGCAGACGAGCGCCAATATCACTGTGGA (SEQ ID NO:156)GATATTGCCTGACGAAGACCCAGAACTGGATAAG GCATTCTCTGTGTCA-3′.

[0576] The mixture was heated in a thermal cycler to 95° C. for 2minutes. Fourteen microliters of 2×hybridization buffer (50% formamide,1.5 M NaCl, 0.04 M NaPO₄, pH 7.2, 5 mM EDTA, 0.2% SDS) were added to theheated probe/cDNA library mixture and incubated at 42° C. for 26 hours.Hybrids between the biotinylated oligo and the circular cDNA wereisolated by diluting the hybridization mixture to 220 microliters in asolution containing 1 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0and adding 125 microliters of streptavidin magnetic beads. This solutionwas incubated at 42° C. for 60 minutes, and mixed every 5 minutes toresuspend the beads. The beads were separated from the solution with amagnet and washed three times in 200 microliters of 0.1×SSPE, 0.1% SDSat 45° C.

[0577] The single stranded cDNAs was release from the biotinylatedoligo/streptavidin magnetic bead complex by adding 50 microliters of 0.1N NaOH and incubating at room temperature for 10 minutes. Sixmicroliters of 3 M sodium acetate was added along with 15 μg of glycogenand the solution was ethanol precipitated with 120 microliters of 100%ethanol. The precipitated DNA was resuspend in 12 μL of TE (10 mMTris-HCl, pH 8.0, 1 mM EDTA, pH 8.0). The single stranded cDNA wasconverted into double strands in a thermal cycler by mixing 5 μL of thecaptured DNA with 1.5 μL of 10 μM of standard SP6 primer for libraries(ATTTAGGTGACACTATAG-3′(SEQ ID NO:157)) in pSPORT 1 and 2, and T7 Sportprimer (5′-TAATACGACTCACTATAGGG-3′ (SEQ ID NO:158)) for libraries inpCMVSPORT, and 1.5 μL of 10×PCR buffer. The mixture was heated to 95° C.for 20 seconds, and then ramped down to 59° C. At this time 15 μL of arepair mix, preheated to 70° C., was added to the DNA (Repair mixcontains 4 μL of 5 mM dNTPs (1.25 mM each), 1.5 μL of 10×PCR buffer,9.25 μL of water, and 0.25 μL of Taq polymerase). The solution wasramped back to 73° C. and incubated for 23 minutes.

[0578] The repaired DNA was ethanol precipitated and resuspended in 10μL of TE. Two μL were electroporated per tube containing 40 μL of E.coli DH12S cells. Three hundred and thirty three μL were plated onto one150 mm plate of LB agar plus 100 μg/mL of ampicillin. After overnightincubation at 37° C., the colonies from all plates were harvested byscraping into 10 mL of LB+50 μg/mL of ampicillin and 2 mL of sterileglycerol.

[0579] The second round of selection was initiated by makingsingle-strand circular DNA from the primary selected library using theabove-described method. The purified single-stranded circular DNA wasthen assayed with gene-specific primers for each of the targetedsequences using standard PCR conditions.

[0580] In the case of Gene 13, the following gene-specific primers(GSPs) were used:

[0581] Primer Set One: left primer 1: 5′-GTGACAATTGCAGCCTCTGA-3′ (SEQ IDNO:151), right primer 1: 5′-AGTGATATTGGCGCTCGTCT-3′ (SEQ ID NO:152);

[0582] Primer Set Two: left primer 2: 5′-CTTCACCTCTGGCAACATCA-3′ (SEQ IDNO:153), right primer 2: 5′-ACTTTTCCCATGAGGCCTTT-3′ (SEQ ID NO:154),

[0583] The hybridization was performed including only those 80 merbiotinylated probes whose targeted sequences had a positive result withthe GSPs. In the case of Gene 13, SEQ ID NO:155 and 156 were used. Theresulting single-stranded circular DNA was converted into double strandsusing the antisense oligo for each target sequence as the repair primer(the sense primer was used for material captured from pSPORT2libraries). The resulting double stranded DNA was electroporated intoDH10B cells and the resulting colonies were inoculated into 96 deep wellblocks. After overnight growth, DNA was prepared and sequentiallyscreened for each of the targeted sequences using the GSPs. The DNA wasalso digested with SalI and NotI restriction enzymes and the insertswere sized by agarose gel electrophoresis.

Example 4 RNA Ligase Protocol for Generating the 5′ or 3′ End Sequencesto Obtain Full-Length GPCR Genes

[0584] Once a GPCR gene/polynucleotide sequence of interest isidentified, several methods are available for the identification of the5′ or 3′ portions of the gene which may not be present in the originalcDNA plasmid. These methods include, but are not limited to, filterprobing, clone enrichment using specific probes and protocols similarand identical to 5′ and 3′RACE. While the full-length gene may bepresent in the library and can be identified by probing, a useful methodfor generating the 5′ or 3′ end is to use the existing sequenceinformation from the original cDNA to generate the missing information.A method similar to 5′RACE is available for generating the missing 5′end of a desired full-length gene. (This method was published byFromont-Racine et al., Nucleic Acids Res., 21(7): 1683-1684 (1993)).

[0585] Briefly, a specific RNA oligonucleotide is ligated to the 5′ endsof a population of RNA, preferably 30, containing full-length gene RNAtranscripts and a primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest, and is used to PCR amplify the 5′ portion of thedesired full length gene which may then be sequenced and used togenerate the full-length gene. This method starts with total RNAisolated from the desired source. PolyA RNA may be used, but is not aprerequisite for this procedure. The RNA preparation is then be treatedwith phosphatase if necessary to eliminate 5′ phosphate groups ondegraded or damaged RNA which may interfere with the later RNA ligasestep. The phosphatase, if used, is then inactivated and the RNA istreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase. This modifiedRNA preparation can then be used as a template for first strand cDNAsynthesis using a gene specific oligonucleotide. The first strandsynthesis reaction can then be used as a template for PCR amplificationof the desired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of theapoptosis related of interest. The resultant product is then sequencedand analyzed to confirm that the 5′ end sequence belongs to the relevantsequence of interest.

Example 5 Signal Transduction Assays

[0586] The activity of GPCRs or homologues thereof, can be measuredusing any assay suitable for the measurement of the activity of a Gprotein-coupled receptor, as commonly known in the art. Signaltransduction activity of a G protein-coupled receptor can be determinedby monitoring intracellular Ca²⁺, cAMP, inositol-1,4,5-triphosphate(IP₃), or 1,2-diacylglycerol (DAG). Assays for the measurement ofintracellular Ca²⁺ are described, for example, in Sakurai et al. (EP 480381). Intracellular IP₃ can be measured using a kit available fromAmersham, Inc. (Arlington Heights, Ill.). A kit for measuringintracellular cAMP is available from Diagnostic Products, Inc. (LosAngeles, Calif.).

[0587] Activation of a G protein-coupled receptor triggers the releaseof Ca²⁺ ions sequestered in the mitochondria, endoplasmic reticulum, andother cytoplasmic vesicles into the cytoplasm. Fluorescent dyes, e.g.,fura-2, can be used to measure the concentration of free cytoplasmicCa²⁺. The ester of fura-2, which is lipophilic and can diffuse acrossthe cell membrane, is added to the culture medium of the host cellswhich recombinantly express GPCRs. Once inside the cell, the fura-2ester is hydrolyzed by cytosolic esterases to its non-lipophilic form,and then the dye cannot diffuse out of the cell. The non-lipophilic formof fura-2 fluoresces when it binds to free Ca²⁺. The fluorescence can bemeasured without lysing the cells at an excitation spectrum of 340 nm or380 nm and at fluorescence spectrum of 500 nm (Sakurai et al., EP 480381).

[0588] Upon activation of a GPCR, the rise of free cytosolic Ca²⁺concentration is preceded by the hydrolysis of phosphatidylinositol4,5-bisphosphate. Hydrolysis of this phospholipid by the phospholipase,phospholipase C, yields 1,2-diacylglycerol (DAG), which remains in themembrane, and water-soluble inositol 1,4,5-triphosphate (IP₃). Bindingof ligands or agonists will increase the concentration of DAG and IP₃.Thus, signal transduction activity can be measured by monitoring theconcentration of these hydrolysis products.

[0589] To measure IP₃ concentration, radioactively-labeled([³H])-inositol is added to the culture medium of host cells expressingGPCRs. The ³H-inositol is taken up by the cells and incorporated intoIP₃. The resulting inositol triphosphate is separated from the mono- anddi-phosphate forms and measured (Sakurai et al., EP 480 381).Alternatively, an inositol 1,4,5-triphosphate assay system (Amersham) iscommercially available for such use. With this system, the supplier(Amersham) provides tritium-labeled inositol 1,4,5-triphosphate and areceptor capable of distinguishing the radioactive inositol from otherinositol phosphates. With these reagents, an effective and accuratecompetition assay can be performed to determine the inositoltriphosphate levels.

[0590] Cyclic AMP levels can be measured according to the methodsdescribed in Gilman et al., Proc. Natl. Acad. Sci, 67:305-312 (1970). Inaddition, a kit for assaying levels of cAMP is available from DiagnosticProducts Corp. (Los Angeles, Calif.).

Example 6 Expression Profiling of Novel Human GPCR Polypeptides

[0591] The same PCR primer pairs used to identify GPCR cDNA clones canbe used to measure the steady state levels of mRNA by quantitative PCR.For example, the PCR primer pairs as set forth in Table 2 herein can beused to measure the steady state levels of the newly described GPCR mRNAby quantitative PCR. In the case of Gene 13, the SEQ ID NO:151 and 152,and/or SEQ ID NO:153 and 154 primers pairs were used for expressionprofiling.

[0592] Briefly, first strand cDNA is made from commercially availablemRNA (Clontech) and subjected to real time quantitative PCR using a PE5700 instrument (Applied Biosystems, Foster City, Calif.) which detectsthe amount of DNA amplified during each cycle by the fluorescent outputof SYBR green, a DNA binding dye specific for double strands. Thespecificity of the primer pair for its target is verified by performinga thermal denaturation profile at the end of the run which provided anindication of the number of different DNA sequences present bydetermining melting Tm. The contribution of contaminating genomic DNA tothe assessment of tissue abundance is controlled for by performing thePCR with first strand made with and without reverse transcriptase. Inall cases, the contribution of material amplified in the no reversetranscriptase controls is expected to be negligible.

[0593] Small variations in the amount of cDNA used in each tube aredetermined by performing a parallel experiment using a primer pair forthe cyclophilin gene, which is expressed in equal amounts in alltissues. These data are used to normalize the data obtained with theprimer pairs. The PCR data are converted into a relative assessment ofthe differences in transcript abundance among the tissues tested.

[0594] As indicated in Table 1, transcripts corresponding to GPCR Gene 4as described herein were found to be expressed in lung; transcriptscorresponding to GPCR Gene 5 as described herein were found to beexpressed in uterus; transcripts corresponding to GPCR Gene 7 asdescribed herein were found to be expressed in skull tumor; transcriptscorresponding to GPCR Gene 9 as described herein were found to beexpressed in cartilage; and transcripts corresponding to GPCR Gene 13 asdescribed herein were found to be expressed in brain.

[0595] Moreover, as indicated in FIG. 41, transcripts corresponding toGene 13 as described herein were found to be expressed to varyingextents in brain, heart, kidney, lung, pancreas, pituitary, smallintestine, spinal cord, testis and thymus.

[0596] As indicated in FIG. 42, transcripts corresponding to Gene 13 asdescribed herein were found to be expressed in the following brain subregions: amygdala, cerebellum, corpus callosum, caudate nucleus,hippocampus, subtantia nigra and thalamus.

Example 7 GPCR Activity

[0597] This example describes another method for screening compoundswhich are GPCR antagonists, and thus inhibit the activation or functionof the GPCR polypeptides of the present invention. The method involvesdetermining inhibition of binding of a labeled ligand, such as dATP,dAMP, or UTP, to cells expressing a novel GPCR on the cell surface, orto cell membranes containing the GPCR.

[0598] Such a method further involves transfecting a eukaryotic cellwith DNA encoding a GPCR polypeptide such that the cell expresses thereceptor on its surface. The cell is then contacted with a potentialantagonist in the presence of a labeled form of a ligand, such as dATP,dAMP, or UTP. The ligand can be labeled, e.g., by radioactivity,fluorescence, chemiluminescence, or any other suitable detectable labelcommonly known in the art. The amount of labeled ligand bound to theexpressed GPCR receptors is measured, e.g., by measuring radioactivityassociated with transfected cells, or membranes from these cells. If thecompound binds to the expressed GPCR, the binding of labeled ligand tothe receptor is inhibited, as determined by a reduction of labeledligand which also binds to the GPCR. This method is called a bindingassay. The above-described technique can also be used to determinebinding of GPCR agonists.

[0599] In a further screening procedure, mammalian cells, for example,but not limited to, CHO, HEK 293, Xenopus oocytes, RBL-2H3, etc., whichare transfected with nucleic acid encoding a novel GPCR, are used toexpress the receptor of interest. The cells are loaded with an indicatordye that produces a fluorescent signal when bound to calcium, and thecells are contacted with a test substance and a receptor agonist, suchas DATP, DAMP, or UTP. Any change in fluorescent signal is measured overa defined period of time using, for example, a fluorescencespectrophotometer or a fluorescence imaging plate reader. A change inthe fluorescence signal pattern generated by the ligand relative tocontrol indicates that a compound is a potential antagonist or agonistfor the receptor.

[0600] In yet another screening procedure, mammalian cells aretransfected with a GPCR-encoding polynucleotide sequence so as toexpress the GPCR of interest. The same cells are also transfected with areporter gene construct that is coupled to/associated with activation ofthe receptor. Nonlimiting examples of suitable reporter gene systemsinclude luciferase or beta-galactosidase regulated by an appropriatepromoter. The engineered cells are contacted with a test substance orcompound and a receptor ligand, such as dATP, dAMP, or UTP, and thesignal produced by the reporter gene is measured after a defined periodof time. The signal can be measured using a luminometer,spectrophotometer, fluorimeter, or other such instrument appropriate forthe specific reporter construct used. Inhibition of the signal generatedby the ligand indicates that a compound is a potential antagonist forthe receptor.

[0601] Another screening technique for determining GPCR antagonists oragonists involves introducing RNA encoding the GPCR polypeptide intocells (e.g., CHO, HEK 293, RBL-2H3 cells, and the like) in which thereceptor is transiently or stably expressed. The receptor cells are thencontacted with a ligand for the GPCR, such as dATP, dAMP, or UTP, and acompound to be screened. Inhibition or activation of the receptor isthen determined by detection of a signal, such as, cAMP, calcium,proton, or other ions.

Example 8 Functional Characterization of the Novel Human GPCR, Gene 13

[0602] The use of mammalian cell reporter assays to demonstratefunctional coupling of known GPCRs has been well documented in theliterature (Gilman, 1987 Ann. Rev. Biochem. 56: 615-649; Boss et al.,1996, J. Biol. Chem., 271: 10429-14032; Alam & Cook, 1990, Anal.Biochem., 188: 245-254; George et al., 1997, J. Neurochem., 69:1278-1285; Selbie & Hill, 1998, TiPs, 19: 87-93; Rees et al., 1999, InMilligan G. (ed.): Signal Transduction: A practical approach, Oxford:Oxford Univ. Press, 171-221). In fact, reporter assays have beensuccessfully used for identifying novel small molecule agonists orantagonists against GPCRs as a class of drug targets (Zlokarnik et al.,1998, Science, 279: 84-88; George et al; Boss et al.; Rees et al.). Insuch reporter assays, a promoter is regulated as a direct consequence ofactivation of specific signal transduction cascades following agonistbinding to a GPCR (Alam & Cook 1990; Selbie & Hill, 1998; Boss et al.;George et al. 1997; Gilman, 1987).

[0603] A number of response element-based reporter systems have beendeveloped that enable the study of GPCR function. These include cAMPresponse element (CRE)-based reporter genes for G alpha i/o, G alphas-coupled GPCRs, Nuclear Factor Activator of Transcription (NFAT)-basedreporters for G alpha q/11-coupled receptors and MAP kinase reportergenes for use in Galpha i/o coupled receptors (Selbie & Hill, 1998; Bosset al. 1996; George et al. 1997; Gilman 1987; Rees et al. 1999).Transcriptional response elements that regulate the expression ofBeta-Lactamase within a CHO K1 cell line (Cho/NFAT-CRE: AuroraBiosciences™) (Zlokarnik et al., 1998) have been implemented tocharacterize the function of the Gene 13 polypeptide of the presentinvention. The system enables demonstration of constitutive G-proteincoupling to endogenous cellular signaling components upon intracellularoverexpression of GPCR receptors. Overexpression has been shown torepresent a physiologically relevant event. For example, it has beenshown that overexpression occurs in nature during metastatic carcinomas,wherein defective expression of the monocyte chemotactic protein 1receptor, CCR2, in macrophages is associated with the incidence of humanovarian carcinoma (Sica, et al., 2000, J. Immunol., 164: 733-8; Salcedoet al., 2000, Blood, 96(1): 34-40). Indeed, it has been shown thatoverproduction of the Beta 2 Adrenergic Receptor in transgenic miceleads to constitutive activation of the receptor signaling pathway suchthat these mice exhibit increased cardiac output (Kypson et al., 1999,Gene Therapy, 6: 1298-1304; Dorn et al., 1999, PNAS, 96: 6400-5). Theseare only a few of the many examples demonstrating constitutiveactivation of GPCRs whereby many of these receptors are likely to be inthe active, R*, conformation (Wess, 1997, FASEB J., 11(5): 346-354).

[0604] A. Materials and Methods:

[0605] DNA Constructs:

[0606] The putative GPCR Gene 13 cDNA may be PCR amplified using PFU™(Stratagene). The primers used in the PCR reaction are specific to theGene 13 polynucleotide and are ordered from Gibco BRL (5 prime primer:5′-CCCAAGCTTATGCAGGCGCTTAACATTACCCCG-3′ (SEQ ID NO:159), 3 prime primer:5′-CGGGATCCTTAATGCCACTGTCTAAAGGAAGA-3′ (SEQ ID NO: 160). The following 3prime primer may be used to add a Flag-tag epitope to the Gene 13polypeptide for immunocytochemistry:5′-CGGGATCCTTACTTGTCGTCGTCGTCCTTGTAGTCCATATGCCCACTGTCTAAAGGAGAATTCTCAAC-3′(SEQ ID NO:161). The product from the PCR reactionmay be isolated from a 0.8% Agarose gel (Invitrogen) and purified usinga Gel Extraction Kit™ from Qiagen.

[0607] The purified product may be then digested overnight along withthe pcDNA3.1 Hygro™ mammalian expression vector from Invitrogen usingthe HindIII and BamHI restriction enzymes (New England Biolabs). Thesedigested products are then purified using the Gel Extraction Kit™ fromQiagen and subsequently ligated to the pcDNA3.1 Hygro™ expression vectorusing a DNA molar ratio of 4 parts insert: 1 vector. All DNAmodification enzymes are purchased from NEB. The ligation may beincubated overnight at 16 degrees Celsius, after which time, onemicroliter of the mix may be used to transform DH5 alpha cloningefficiency competent E. coli™ (Gibco BRL). A detailed description of thepcDNA3.1 Hygro™ mammalian expression vector is available from Invitrogen(1600 Faraday Avenue, P.O. Box 6482, Carlsbad, Calif. 92008). Theplasmid DNA from the ampicillin resistant clones are isolated using theWizard DNA Miniprep System™ from Promega. Positive clones are thenconfirmed and scaled up for purification using the Qiagen Maxiprep™plasmid DNA purification kit.

[0608] B. Cell Line Generation:

[0609] The pcDNA3.1 hygro vector containing the GPCR Gene 13 cDNA areused to transfect Cho/NFAT-CRE (Aurora Biosciences) cells usingLipofectamine 2000™ according to the manufacturers specifications (GibcoBRL). Two days later, the cells are split 1:3 into selective media (DMEM11056, 600 ug/ml Hygromycin, 200 ug/ml Zeocin, 10% FBS). All cellculture reagents are purchased from Gibco BRL-Invitrogen.

[0610] The Cho/NFAT-CRE cell lines, transiently or stably transfectedwith the Gene 13 GPCR, are analyzed using the FACS Vantage SE™ (BD),fluorescence microscopy (Nikon), and the LJL Analyst™ (MolecularDevices). In this system, changes in real-time gene expression, as aconsequence of constitutive G-protein coupling of the Gene 13 GPCR, isexamined by analyzing the fluorescence emission of the transformed cellsat 447 nm and 518 nm. The changes in gene expression can be visualizedusing Beta-Lactamase as a reporter, that, when induced by theappropriate signaling cascade, hydrolyzes an intracellularly loaded,membrane-permeant ester,Cephalosporin-Coumarin-Fluorescein-2/Acetoxymethyl™ (CCF2/AM™ AuroraBiosciences; Zlokarnik, et al., 1998). The CCF2/AM™ substrate is a7-hydroxycoumarin cephalosporin with a fluorescein attached through astable thioether linkage. Induced expression of the Beta-Lactamaseenzyme is readily apparent since each enzyme molecule produced iscapable of changing the fluorescence of many CCF2/AM™ substratemolecules. A schematic of this cell based system is shown in FIG. 9.

[0611] In summary, CCF2/AM™ is a membrane permeant,intracellularly-trapped, fluorescent substrate with a cephalosporin corethat links a 7-hydroxycoumarin to a fluorescein. For the intactmolecule, excitation of the coumarin at 409 nm results in FluorescenceResonance Energy Transfer (FRET) to the fluorescein which emits greenlight at 518 nm. Production of active Beta-Lactamase results in cleavageof the Beta-Lactam ring, leading to disruption of FRET, and excitationof the coumarin only-thus giving rise to blue fluorescent emission at447 nm.

[0612] Fluorescent emissions are detected using a Nikon-TE300 microscopeequipped with an excitation filter (D405/10X-25), dichroic reflector(430DCLP), and a barrier filter for dual DAPI/FITC (510 nM) to visuallycapture changes in Beta-Lactamase expression. The FACS Vantage SE isequipped with a Coherent Enterprise II Argon Laser and a Coherent 302CKrypton laser. In flow cytometry, UV excitation at 351-364 nm from theArgon Laser or violet excitation at 407 nm from the Krypton laser areused. The optical filters on the FACS Vantage SE are HQ460/50m andHQ535/40m bandpass separated by a 490 dichroic mirror.

[0613] Prior to analyzing the fluorescent emissions from the cell linesas described above, the cells are loaded with the CCF2/AM substrate. A6×CCF2/AM loading buffer may be prepared whereby 1 mM CCF2/AM (AuroraBiosciences) may be dissolved in 100% DMSO (Sigma). 12 ul of this stocksolution may be added to 60 ul of 100 mg/ml Pluronic F127 (Sigma) inDMSO containing 0.1% Acetic Acid (Sigma). This solution may be addedwhile vortexing to 1 mL of Sort Buffer (PBS minus calcium andmagnesium-Gibco-25 mM HEPES-Gibco-pH 7.4, 0.1% BSA). Cells are placed inserum-free media and the 6×CCF2/AM may be added to a final concentrationof 1×. The cells are then loaded at room temperature for one to twohours, and then subjected to fluorescent emission analysis as describedherein. Additional details relative to the cell loading methods and/orinstrument settings may be found by reference to the followingpublications: see Zlokarnik, et al., 1998; Whitney et al., 1998, NatureBiotech. 16: 1329-1333; and BD Biosciences, 1999, FACS Vantage SETraining Manual.

[0614] C. Immunocytochemistry:

[0615] The cell lines transfected and selected for expression ofFlag-epitope tagged GPCRs are analyzed by immunocytochemistry. The cellsare plated at 1×10^ 3 in each well of a glass slide (VWR). The cells arerinsed with PBS followed by acid fixation for 30 minutes at roomtemperature using a mixture of 5% Glacial Acetic Acid/90% ETOH. Thecells are then blocked in 2% BSA and 0.1% Triton in PBS, incubated for 2h at room temperature or overnight at 4° C. A monoclonal anti-Flag FITCantibody may be diluted at 1:50 in blocking solution and incubated withthe cells for 2 h at room temperature. Cells are then washed three timeswith 0.1% Triton in PBS for five minutes. The slides are overlayed withmounting media dropwise with Biomedia-Gel Mount™ (Biomedia; ContainingAnti-Quenching Agent). Cells are examined at 10× magnification using theNikon TE300 equiped with FITC filter (535 nm).

[0616] D. Demonstration of Cell Surface Expression:

[0617] Gene 13 may be tagged at the C-terminus using the Flag epitopeand inserted into the pcDNA3.1 hygro™ expression vector, as describedherein.

[0618] Immunocytochemistry of Cho Nfat-CRE cell lines transfected withthe Flag-tagged Gene 13 construct with FITC conjugated Anti Flagmonoclonal antibody demonstrated that Gene 13 is indeed a cell surfacereceptor. The immunocytochemistry also confirmed expression of the Gene13 in the Cho Nfat-CRE cell lines. Briefly, Cho Nfat-CRE cell lines aretransfected with pcDNA3.1 TM hygro™/Gene 13-Flag vector, fixed with 70%methanol, and permeablized with 0.1% TritonX100. The cells are thenblocked with 1% Serum and incubated with a FITC conjugated Anti Flagmonoclonal antibody at 1:50 dilution in PBS-Triton. The cells are thenwashed several times with PBS-Triton, overlayed with mounting solution,and fluorescent images are captured. The control cell line,non-transfected ChoNfat CRE cell line, exhibited no detectablebackground fluorescence. Plasma membrane localization would beconsistent with Gene 13 representing a 7 transmembrane domain containingGPCR.

[0619] E. Screening Paradigm

[0620] The Aurora Beta-Lactamase technology provides a clear path foridentifying agonists and antagonists of the Gene 13 polypeptide. Celllines that exhibit a range of constitutive coupling activity may beidentified by sorting through Gene 13 transfected cell lines using theFACS Vantage SE (see FIG. 10). For example, cell lines that exhibit anintermediate coupling response, using the LJL analyst, would provide theopportunity to screen, indirectly, for both agonists and antogonists ofGene 13 by looking for inhibitors that block the beta lactamaseresponse, or agonists that increase the beta lactamase response. Asdescribed herein, modulating the expression level of beta lactamasedirectly correlates with the level of cleaved CCR2 substrate. Forexample, this screening paradigm has been shown to work for theidentification of modulators of a known GPCR, 5HT6, that couples throughAdenylate Cyclase, in addition to, the identification of modulators ofthe 5HT2c GPCR, that couples through changes in [Ca²⁺]i. Gene 13modulator screens may be carried out using a variety of high throughputmethods known in the art, though preferably using the fully automatedAurora UHTSS system.

[0621] In preferred embodiments, the Gene 13 transfected Cho Nfat-CREcell lines are useful for the identification of agonists and antagonistsof the Gene 13 polypeptide. Representative uses of these cell lineswould be their inclusion in a method of identifying Gene 13 agonists andantagonists. Preferably, the cell lines are useful in a method foridentifying a compound that modulates the biological activity of theGene 13 polypeptide, comprising the steps of (a) combining a candidatemodulator compound with a host cell expressing the Gene 13 polypeptidehaving the sequence as set forth in SEQ ID NO:2; and (b) measuring aneffect of the candidate modulator compound on the activity of theexpressed Gene 13 polypeptide. Representative vectors expressing theGene 13 polypeptide are referenced herein (e.g., pcDNA3.1 hygro™) orotherwise known in the art.

[0622] The cell lines are also useful in a method of screening for acompound that is capable of modulating the biological activity of Gene13 polypeptide, comprising the steps of: (a) determining the biologicalactivity of the Gene 13 polypeptide in the absence of a modulatorcompound; (b) contacting a host cell expression the Gene 13 polypeptidewith the modulator compound; and (c) determining the biological activityof the Gene 13 polypeptide in the presence of the modulator compound;wherein a difference between the activity of the Gene 13 polypeptide inthe presence of the modulator compound and in the absence of themodulator compound indicates a modulating effect of the compound.Additional uses for these cell lines are described herein or otherwiseknown in the art.

[0623] The present invention is meant to encompass the application ofthe same coupling assay to the functional characterization of Gene 7,and 10, as well.

Example 9 Method of Creating N- and C-Terminal Deletion MutantsCorresponding to the Gene 13 Polypeptide of the Present Invention

[0624] As described elsewhere herein, the present invention encompassesthe creation of N- and C-terminal deletion mutants, in addition to anycombination of N- and C-terminal deletions thereof, corresponding to theGene 13 polypeptide of the present invention. A number of methods areavailable to one skilled in the art for creating such mutants. Suchmethods may include a combination of PCR amplification and gene cloningmethodology. Although one of skill in the art of molecular biology,through the use of the teachings provided or referenced herein, and/orotherwise known in the art as standard methods, could readily createeach deletion mutant of the present invention, exemplary methods aredescribed below.

[0625] Briefly, using the isolated cDNA clone encoding the full-lengthGene 13 polypeptide sequence (as described in Examples 3 and 4, forexample), appropriate primers of about 15-25 nucleotides derived fromthe desired 5′ and 3′ positions of SEQ ID NO:13 may be designed to PCRamplify, and subsequently clone, the intended N- and/or C-terminaldeletion mutant. Such primers could comprise, for example, aninititation and stop codon for the 5′ and 3′ primer, respectively. Suchprimers may also comprise restriction sites to facilitate cloning of thedeletion mutant post amplification. Moreover, the primers may compriseadditional sequences, such as, for example, flag-tag sequences, kozaksequences, or other sequences discussed and/or referenced herein.

[0626] For example, in the case of the L4 to Y294 N-terminal deletionmutant, the following primers in Table 5 could be used to amplify a cDNAfragment corresponding to this deletion mutant: TABLE 5 5′ Primer5′-gcagca gcggccgc ctcttttcaaaaagttgttccttgg-3′ (SEQ ID NO:162) NotI 3′Primer 5′-gcagca gtcgac ataataataacacactaagggatac-3′ (SEQ ID NO:163)SalI

[0627] For example, in the case of the M1 to P288 C-terminal deletionmutant, the following primers in Table 6 could be used to amplify a cDNAfragment corresponding to this deletion mutant: TABLE 6 5′ Primer5′-gcagca gcggccgc atggaaggactcttttcaaaaag-3′ (SEQ ID NO:164) NotI 3′Primer 5′-gcagca gtcgac gggatacttacttgaacgactctg-3′ (SEQ ID NO:165) SalI

[0628] Representative PCR amplification conditions are provided below,although the skilled artisan would appreciate that other conditions maybe required for efficient amplification. A 100 ul PCR reaction mixturemay be prepared using 10 ng of the template DNA (cDNA clone of Gene 13),200 uM 4dNTPs, 1 uM primers, 0.25U Taq DNA polymerase (PE), and standardTaq DNA polymerase buffer. Typical PCR cycling condition are as follows:20-25 cycles of: (45 sec, 93 degrees; 2 min, 50 degrees; 2 min, 72degrees) and 1 cycle of: (10 min, 72 degrees). After the final extensionstep of PCR, 5U Klenow Fragment may be added and incubated for 15 min at30 degrees.

[0629] Upon digestion of the fragment with the NotI and SalI restrictionenzymes, the fragment could be cloned into an appropriate expressionand/or cloning vector which has been similarly digested (e.g., pSport1,among others). The skilled artisan would appreciate that other plasmidscould be equally substituted, and may be desirable in certaincircumstances. The digested fragment and vector are then ligated using aDNA ligase, and then used to transform competent E.coli cells usingmethods provided herein and/or otherwise known in the art.

[0630] The 5′ primer sequence for amplifying any additional N-terminaldeletion mutants may be determined by reference to the followingformula: (S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to thenucleotide position of the initiating start codon of the Gene 13 gene(SEQ ID NO:13), and ‘X’ is equal to the most N-terminal amino acid ofthe intended N-terminal deletion mutant. The first term will provide thestart 5′ nucleotide position of the 5′ primer, while the second termwill provide the end 3′ nucleotide position of the 5′ primercorresponding to sense strand of SEQ ID NO:13. Once the correspondingnucleotide positions of the primer are determined, the final nucleotidesequence may be created by the addition of applicable restriction sitesequences to the 5′ end of the sequence, for example. As referencedherein, the addition of other sequences to the 5′ primer may be desiredin certain circumstances (e.g., kozak sequences, etc.).

[0631] The 3′ primer sequence for amplifying any additional N-terminaldeletion mutants may be determined by reference to the followingformula: (S+(X*3)) to ((S+(X*3))−25), wherein ‘S’ is equal to thenucleotide position of the initiating start codon of the Gene 13 gene(SEQ ID NO:13), and ‘X’ is equal to the most C-terminal amino acid ofthe intended N-terminal deletion mutant. The first term will provide thestart 5′ nucleotide position of the 3′ primer, while the second termwill provide the end 3′ nucleotide position of the 3′ primercorresponding to the anti-sense strand of SEQ ID NO:1. Once thecorresponding nucleotide positions of the primer are determined, thefinal nucleotide sequence may be created by the addition of applicablerestriction site sequences to the 5′ end of the sequence, for example.As referenced herein, the addition of other sequences to the 3′ primermay be desired in certain circumstances (e.g., stop codon sequences,etc.). The skilled artisan would appreciate that modifications of theabove nucleotide positions may be necessary for optimizing PCRamplification.

[0632] The same general formulas provided above may be used inidentifying the 5′ and 3′ primer sequences for amplifying any C-terminaldeletion mutant of the present invention. Moreover, the same generalformulas provided above may be used in identifying the 5′ and 3′ primersequences for amplifying any combination of N-terminal and C-terminaldeletion mutant of the present invention. The skilled artisan wouldappreciate that modifications of the above nucleotide positions may benecessary for optimizing PCR amplification.

[0633] Moreover, the invention encompasses the application of the sameformulas and methods to the creation of any N—, or C-terminal deletionmutant, or any combination thereof, of Gene 7 and/or 10.

Example 10 Method of Assessing the Expression Profile of the Novel Gene13 Polypeptides of the Present Invention Using Expanded MRNA Tissue andCell Sources

[0634] Total RNA from tissues was isolated using the TriZol protocol(Invitrogen) and quantified by determining its absorbance at 260 nM. Anassessment of the 18s and 28s ribosomal RNA bands was made by denaturinggel electrophoresis to determine RNA integrity.

[0635] The specific sequence to be measured was aligned with relatedgenes found in GenBank to identity regions of significant sequencedivergence to maximize primer and probe specificity. Gene-specificprimers and probes were designed using the ABI primer express softwareto amplify small amplicons (150 base pairs or less) to maximize thelikelihood that the primers function at 100% efficiency. Allprimer/probe sequences were searched against Public Genbank databases toensure target specificity. Primers and probes were obtained from ABI.

[0636] For Gene 13, the primer probe sequences were as follows

[0637] Forward Primer 5′-GCAGACGAGCGCCAATATC-3′ (SEQ ID NO:166)

[0638] Reverse Primer 5′-GACACAGAGAATGCCTTATCCAGTT-3′ (SEQ ID NO: 167)

[0639] TaqMan Probe5′-TGGGTCTTCGTCAGGCAATATCTCCACA-3′ (SEQ ID NO: 168)

[0640] I. DNA Contamination

[0641] To access the level of contaminating genomic DNA in the RNA, theRNA was divided into 2 aliquots and one half was treated with Rnase-freeDnase (Invitrogen). Samples from both the Dnase-treated and non-treatedwere then subjected to reverse transcription reactions with (RT+) andwithout (RT−) the presence of reverse transcriptase. TaqMan assays werecarried out with gene-specific primers (see above) and the contributionof genomic DNA to the signal detected was evaluated by comparing thethreshold cycles obtained with the RT+/RT− non-Dnase treated RNA to thaton the RT+/RT− Dnase treated RNA. The amount of signal contributed bygenomic DNA in the Dnased RT− RNA must be less that 10% of that obtainedwith Dnased RT+ RNA. If not the RNA was not used in actual experiments.

[0642] II. Reverse Transcription Reaction and Sequence Detection

[0643] 100 ng of Dnase-treated total RNA was annealed to 2.5 μM of therespective gene-specific reverse primer in the presence of 5.5 mMMagnesium Chloride by heating the sample to 72° C. for 2 min and thencooling to 55° C. for 30 min. 1.25 U/μl of MuLv reverse transcriptaseand 500 μM of each dNTP was added to the reaction and the tube wasincubated at 37° C. for 30 min. The sample was then heated to 90° C. for5 min to denature enzyme.

[0644] Quantitative sequence detection was carried out on an ABI PRISM7700 by adding to the reverse transcribed reaction 2.5 μM forward andreverse primers, 2.0 μM of the TaqMan probe, 500 μM of each dNTP, bufferand 5U AmpliTaq Gold™. The PCR reaction was then held at 94° C. for 12min, followed by 40 cycles of 94° C. for 15 sec and 60° C. for 30 sec.

[0645] III. Data Handling

[0646] The threshold cycle (Ct) of the lowest expressing tissue (thehighest Ct value) was used as the baseline of expression and all othertissues were expressed as the relative abundance to that tissue bycalculating the difference in Ct value between the baseline and theother tissues and using it as the exponent in 2^((ΔCt))

[0647] The expanded expression profile of the Gene 13 polypeptide isprovided in FIG. 43 and described elsewhere herein.

Example 11 Method of Enhancing the Biological Activity/FunctionalCharacteristics of Invention Through Molecular Evolution.

[0648] Although many of the most biologically active proteins known arehighly effective for their specified function in an organism, they oftenpossess characteristics that make them undesirable for transgenic,therapeutic, pharmaceutical, and/or industrial applications. Among thesetraits, a short physiological half-life is the most prominent problem,and is present either at the level of the protein, or the level of theproteins mRNA. The ability to extend the half-life, for example, wouldbe particularly important for a proteins use in gene therapy, transgenicanimal production, the bioprocess production and purification of theprotein, and use of the protein as a chemical modulator among others.Therefore, there is a need to identify novel variants of isolatedproteins possessing characteristics which enhance their application as atherapeutic for treating diseases of animal origin, in addition to theproteins applicability to common industrial and pharmaceuticalapplications.

[0649] Thus, one aspect of the present invention relates to the abilityto enhance specific characteristics of invention through directedmolecular evolution. Such an enhancement may, in a non-limiting example,benefit the inventions utility as an essential component in a kit, theinventions physical attributes such as its solubility, structure, orcodon optimization, the inventions specific biological activity,including any associated enzymatic activity, the proteins enzymekinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity,protein-DNA binding activity, antagonist/inhibitory activity (includingdirect or indirect interaction), agonist activity (including direct orindirect interaction), the proteins antigenicity (e.g., where it wouldbe desirable to either increase or decrease the antigenic potential ofthe protein), the immunogenicity of the protein, the ability of theprotein to form dimers, trimers, or multimers with either itself orother proteins, the antigenic efficacy of the invention, including itssubsequent use a preventative treatment for disease or disease states,or as an effector for targeting diseased genes. Moreover, the ability toenhance specific characteristics of a protein may also be applicable tochanging the characterized activity of an enzyme to an activitycompletely unrelated to its initially characterized activity. Otherdesirable enhancements of the invention would be specific to eachindividual protein, and would thus be well known in the art andcontemplated by the present invention.

[0650] For example, an engineered G-protein coupled receptor may beconstitutively active upon binding of its cognate ligand. Alternatively,an engineered G-protein coupled receptor may be constitutively active inthe absence of ligand binding. In yet another example, an engineeredGPCR may be capable of being activated with less than all of theregulatory factors and/or conditions typically required for GPCRactivation (e.g., ligand binding, phosphorylation, conformationalchanges, etc.). Such GPCRs would be useful in screens to identify GPCRmodulators, among other uses described herein.

[0651] Directed evolution is comprised of several steps. The first stepis to establish a library of variants for the gene or protein ofinterest. The most important step is to then select for those variantsthat entail the activity you wish to identify. The design of the screenis essential since your screen should be selective enough to eliminatenon-useful variants, but not so stringent as to eliminate all variants.The last step is then to repeat the above steps using the best variantfrom the previous screen. Each successive cycle, can then be tailored asnecessary, such as increasing the stringency of the screen, for example.

[0652] Over the years, there have been a number of methods developed tointroduce mutations into macromolecules. Some of these methods include,random mutagenesis, “error-prone” PCR, chemical mutagenesis,site-directed mutagenesis, and other methods well known in the art (fora comprehensive listing of current mutagenesis methods, see Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Typically, such methods have been used, forexample, as tools for identifying the core functional region(s) of aprotein or the function of specific domains of a protein (if amulti-domain protein). However, such methods have more recently beenapplied to the identification of macromolecule variants with specific orenhanced characteristics.

[0653] Random mutagenesis has been the most widely recognized method todate.

[0654] Typically, this has been carried out either through the use of“error-prone” PCR (as described in Moore, J., et al, NatureBiotechnology 14:458, (1996), or through the application of randomizedsynthetic oligonucleotides corresponding to specific regions of interest(as descibed by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), andHill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approacheshave limits to the level of mutagenesis that can be obtained. However,either approach enables the investigator to effectively control the rateof mutagenesis. This is particularly important considering the fact thatmutations beneficial to the activity of the enzyme are fairly rare. Infact, using too high a level of mutagenesis may counter or inhibit thedesired benefit of a useful mutation.

[0655] While both of the aforementioned methods are effective forcreating randomized pools of macromolecule variants, a third method,termed “DNA Shuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747,(1994)) has recently been elucidated. DNA shuffling has also beenreferred to as “directed molecular evolution”, “exon-shuffling”,“directed enzyme evolution”, “in vitro evolution”, and “artificialevolution”. Such reference terms are known in the art and areencompassed by the invention. This new, preferred, method apparentlyovercomes the limitations of the previous methods in that it not onlypropagates positive traits, but simultaneously eliminates negativetraits in the resulting progeny.

[0656] DNA shuffling accomplishes this task by combining the principalof in vitro recombination, along with the method of “error-prone” PCR.In effect, you begin with a randomly digested pool of small fragments ofyour gene, created by Dnase I digestion, and then introduce said randomfragments into an “error-prone” PCR assembly reaction. During the PCRreaction, the randomly sized DNA fragments not only hybridize to theircognate strand, but also may hybridize to other DNA fragmentscorresponding to different regions of the polynucleotide ofinterest—regions not typically accessible via hybridization of theentire polynucleotide. Moreover, since the PCR assembly reactionutilizes “error-prone” PCR reaction conditions, random mutations areintroduced during the DNA synthesis step of the PCR reaction for all ofthe fragments -further diversifying the potential hybridation sitesduring the annealing step of the reaction.

[0657] A variety of reaction conditions could be utilized to carry-outthe DNA shuffling reaction. However, specific reaction conditions forDNA shuffling are provided, for example, in PNAS, 91:10747, (1994).Briefly:

[0658] Prepare the DNA substrate to be subjected to the DNA shufflingreaction.

[0659] Preparation may be in the form of simply purifying the DNA fromcontaminating cellular material, chemicals, buffers, oligonucleotideprimers, deoxynucleotides, RNAs, etc., and may entail the use of DNApurification kits as those provided by Qiagen, Inc., or by the Promega,Corp., for example.

[0660] Once the DNA substrate has been purified, it would be subjectedto Dnase I digestion. About 2-4 ug of the DNA substrate(s) would bedigested with 0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mMTris-HCL, pH 7.4/1 mM MgCl2 for 10-20 min. at room temperature. Theresulting fragments of 10-50 bp could then be purified by running themthrough a 2% low-melting point agarose gel by electrophoresis onto DE81ion-exchange paper (Whatman) or could be purified using Microconconcentrators (Amicon) of the appropriate molecular weight cuttoff, orcould use oligonucleotide purification columns (Qiagen), in addition toother methods known in the art. If using DE81 ion-exchange paper, the10-50 bp fragments could be eluted from said paper using 1M NaCL,followed by ethanol precipitation.

[0661] The resulting purified fragments would then be subjected to a PCRassembly reaction by re-suspension in a PCR mixture containing: 2 mM ofeach dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris.HCL, pH 9.0, and 0.1%Triton X-100, at a final fragment concentration of 10-30 ng/ul. Noprimers are added at this point. Taq DNA polymerase (Promega) would beused at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 Cfor 60s; 94 C for 30s, 50-55 C for 30s, and 72 C for 30s using 30-45cycles, followed by 72 C for 5 min using an MJ Research (Cambridge,Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a1:40 dilution of the resulting primerless product would then beintroduced into a PCR mixture (using the same buffer mixture used forthe assembly reaction) containing 0.8 um of each primer and subjectingthis mixture to 15 cycles of PCR (using 94 C for 30s, 50 C for 30s, and72 C for 30s). The referred primers would be primers corresponding tothe nucleic acid sequences of the polynucleotide(s) utilized in theshuffling reaction. Said primers could consist of modified nucleic acidbase pairs using methods known in the art and referred to else whereherein, or could contain additional sequences (i.e., for addingrestriction sites, mutating specific base-pairs, etc.).

[0662] The resulting shuffled, assembled, and amplified product can bepurified using methods well known in the art (e.g., Qiagen PCRpurification kits) and then subsequently cloned using appropriaterestriction enzymes.

[0663] Although a number of variations of DNA shuffling have beenpublished to date, such variations would be obvious to the skilledartisan and are encompassed by the invention. The DNA shuffling methodcan also be tailered to the desired level of mutagenesis using themethods described by Zhao, et al. (Nucl Acid Res., 25(6):1307-1308,(1997).

[0664] As described above, once the randomized pool has been created, itcan then be subjected to a specific screen to identify the variantpossessing the desired characteristic(s). Once the variant has beenidentified, DNA corresponding to the variant could then be used as theDNA substrate for initiating another round of DNA shuffling. This cycleof shuffling, selecting the optimized variant of interest, and thenre-shuffling, can be repeated until the ultimate variant is obtained.Examples of model screens applied to identify variants created using DNAshuffling technology may be found in the following publications: J. C.,Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al.,Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat.Biotech., 15:436-438, (1997).

[0665] DNA shuffling has several advantages. First, it makes use ofbeneficial mutations. When combined with screening, DNA shuffling allowsthe discovery of the best mutational combinations and does not assumethat the best combination contains all the mutations in a population.Secondly, recombination occurs simultaneously with point mutagenesis. Aneffect of forcing DNA polymerase to synthesize full-length genes fromthe small fragment DNA pool is a background mutagenesis rate. Incombination with a stringent selection method, enzymatic activity hasbeen evolved up to 16000 fold increase over the wild-type form of theenzyme. In essence, the background mutagenesis yielded the geneticvariability on which recombination acted to enhance the activity.

[0666] A third feature of recombination is that it can be used to removedeleterious mutations. As discussed above, during the process of therandomization, for every one beneficial mutation, there may be at leastone or more neutral or inhibitory mutations. Such mutations can beremoved by including in the assembly reaction an excess of the wild-typerandom-size fragments, in addition to the random-size fragments of theselected mutant from the previous selection. During the next selection,some of the most active variants of thepolynucleotide/polypeptide/enzyme, should have lost the inhibitorymutations.

[0667] Finally, recombination enables parallel processing. Thisrepresents a significant advantage since there are likely multiplecharacteristics that would make a protein more desirable (e.g.solubility, activity, etc.). Since it is increasingly difficult toscreen for more than one desirable trait at a time, other methods ofmolecular evolution tend to be inhibitory. However, using recombination,it would be possible to combine the randomized fragments of the bestrepresentative variants for the various traits, and then select formultiple properties at once.

[0668] DNA shuffling can also be applied to the polynucleotides andpolypeptides of the present invention to decrease their immunogenicityin a specified host, particularly if the polynucleotides andpolypeptides provide a therapeutic use. For example, a particularvariant of the present invention may be created and isolated using DNAshuffling technology. Such a variant may have all of the desiredcharacteristics, though may be highly immunogenic in a host due to itsnovel intrinsic structure. Specifically, the desired characteristic maycause the polypeptide to have a non-native structure which could nolonger be recognized as a “self” molecule, but rather as a “foreign”,and thus activate a host immune response directed against the novelvariant. Such a limitation can be overcome, for example, by including acopy of the gene sequence for a xenobiotic ortholog of the nativeprotein in with the gene sequence of the novel variant gene in one ormore cycles of DNA shuffling. The molar ratio of the ortholog and novelvariant DNAs could be varied accordingly. Ideally, the resulting hybridvariant identified would contain at least some of the coding sequencewhich enabled the xenobiotic protein to evade the host immune system,and additionally, the coding sequence of the original novel varient thatprovided the desired characteristics.

[0669] Likewise, the invention encompasses the application of DNAshuffling technology to the evolution of polynucletotides andpolypeptides of the invention, wherein one or more cycles of DNAshuffling include, in addition to the gene template DNA,oligonucleotides coding for known allelic sequences, optimized codonsequences, known variant sequences, known polynucleotide polymorphismsequences, known ortholog sequences, known homolog sequences, additionalhomologous sequences, additional non-homologous sequences, sequencesfrom another species, and any number and combination of the above.

[0670] In addition to the described methods above, there are a number ofrelated methods that may also be applicable, or desirable in certaincases. Representative among these are the methods discussed in PCTapplications WO 98/31700, and WO 98/32845, which are hereby incorporatedby reference. Furthermore, related methods can also be applied to thepolynucleotide sequences of the present invention in order to evolveinvention for creating ideal variants for use in gene therapy, proteinengineering, evolution of whole cells containing the variant, or in theevolution of entire enzyme pathways containing polynucleotides of theinvention as described in PCT applications WO 98/13485, WO 98/13487, WO98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech.,15:436-438, (1997), respectively.

[0671] Additional methods of applying “DNA Shuffling” technology to thepolynucleotides and polypeptides of the present invention, includingtheir proposed applications, may be found in U.S. Pat. No. 5,605,793;PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCTApplication No. WO 97/35966; and PCT Application No. WO 98/42832; PCTApplication No. The forgoing are hereby incorporated in their entiretyherein for all purposes.

Example 12 Method Of Isolating Antibody Fragments Directed Against Genes1-13 from a Library of scFvs.

[0672] Naturally occurring V-genes isolated from human PBLs areconstructed into a library of antibody fragments which containreactivities against Genes 1-13 to which the donor may or may not havebeen exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein byreference in its entirety).

[0673] Rescue of the Library. A library of scFvs is constructed from theRNA of human PBLs as described in PCT publication WO 92/01047. To rescuephage displaying antibody fragments, approximately 109 E. coli harboringthe phagemid are used to inoculate 50 ml of 2×TY containing 1% glucoseand 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8with shaking. Five ml of this culture is used to inoculate 50 ml of2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

[0674] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)displaying antibody fragments have a greater avidity of binding toantigen. Infectious M13 delta gene III particles are made by growing thehelper phage in cells harboring a pUC19 derivative supplying the wildtype gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37° C. without shaking and then for a furtherhour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μgampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 μm filter (MinisartNML; Sartorius) to give a final concentration of approximately 1013transducing units/ml (ampicillin-resistant clones).

[0675] Panning of the Library. Immunotubes (Nunc) are coated overnightin PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[0676] Characterization of Binders. Eluted phage from the 3rd and 4throunds of selection are used to infect E. coli HB 2151 and soluble scFvis produced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing. These ELISApositive clones may also be further characterized by techniques known inthe art, such as, for example, epitope mapping, binding affinity,receptor signal transduction, ability to block or competitively inhibitantibody/antigen binding, and competitive agonistic or antagonisticactivity.

[0677] Moreover, in another preferred method, the antibodies directedagainst the polypeptides of the present invention may be produced inplants. Specific methods are disclosed in U.S. Pat. Nos. 5,959,177, and6,080,560, which are hereby incorporated in their entirety herein. Themethods not only describe methods of expressing antibodies, but also themeans of assembling foreign multimeric proteins in plants (i.e.,antibodies, etc,), and the subsequent secretion of such antibodies fromthe plant.

[0678] The contents of all patents, patent applications, published PCTapplications and articles, books, references, reference manuals,abstracts and internet websites cited herein are hereby incorporated byreference in their entirety to more fully describe the state of the artto which the invention pertains.

[0679] As various changes can be made in the above-described subjectmatter without departing from the scope and spirit of the presentinvention, it is intended that all subject matter contained in the abovedescription, or defined in the appended claims, be interpreted asdescriptive and illustrative of the present invention. Manymodifications and variations of the present invention are possible inlight of the above teachings. TABLE 1 Novel G-Protein Coupled Receptorsof the Present Invention NUCLEIC ACID GPCR CLONE ID/ FUNCTIONAL TISSUESEQ ID NO./FIG. ENCODED AMINO ACID GENENO. BAC ID ANNOTATION EXPRESSIONNO. SEQ ID NO./FIG. NO. 1 1462859.1 Sensory GPCR Fetal Lung; SEQ IDNO:1; SEQ ID NO:14; Testis; B cells 2 1102336.1 Sensory GPCR ND SEQ IDNO:2; SEQ ID NO:15; 3 BAC:NT_024210 Sensory GPCR ND SEQ ID NO:3; SEQ IDNO:16; 4 27534.1 Sensory GPCR Lung SEQ ID NO:4; SEQ ID NO:17; 5BAC:AC018630 Sensory GPCR Uterus SEQ ID NO:5; SEQ ID NO:18; 6BAC:AC021089 Chemokine ND SEQ ID NO:6; SEQ ID NO:19; GPCR 7 338589.1Human orphan Skull tumor SEQ ID NO:7; SEQ ID NO:20; GPCR FIGS. 13A/13BAAY90761 8 7474790CB1 Human orphan ND SEQ ID NO:8; SEQ ID NO:21; GPCRP1_312546 FIGS. 15A/15B 9 356272.1 Sensory GPCR, Cartilage SEQ ID NO:9;SEQ ID NO:22; P1_314986 10  BAC:AC084434 Sensory GPCR, ND SEQ ID NO:10;SEQ ID NO:23; P2_94452 11  CR_1449607 Sensory GPCR, ND SEQ ID NO:11; SEQID NO:24; P2_94474 FIGS. 21A/21B 12  7474816CB1 Sensory GPCR, ND SEQ IDNO:12; SEQ ID NO:25; P2_96621 13  1137487.1 Very Large Brain SEQ IDNO:13; SEQ ID NO:26; GPCR, P1_404650

[0680] TABLE 2 Predicted Primers for Novel G-Protein Coupled ReceptorsGPCR LEFT PRIMER RIGHT PRIMER  1 tcacggctcccaaatctatctgtgcatcacagcaatcaga (SEQ ID NO:27) (SEQ ID NO:28)  2tctgtaagcagggtgctgtg acaatgaggccgtaggacac (SEQ ID NO:29) (SEQ ID NO:30) 3 tctcaccctcaccaccctac cagcagcaaatgatggctaa (SEQ ID NO:31) (SEQ IDNO:32)  4 acctgcaccaccactctacc gggaaaggaatcatcagcaa (SEQ ID NO:33) (SEQID NO:34)  5 taaattccattgagcgggtc agcaagccagttgctgaaat (SEQ ID NO:35)(SEQ ID NO:36)  6a gctggtacacatcatgtccg acaggttgcttctggcactt 1st (SEQ IDNO:37) (SEQ ID NO:38) primer  6b cctccatctttgccacacttggcacttgaagaaagccttg 2nd (SEQ ID NO:39) (SEQ ID NO:40) primer  7cagctcctgtaggcatctcc caccagtctgatgacccctt (SEQ ID NO:41) (SEQ ID NO:42) 8 tcagtgaggatgacgtcgag gcaggaagaaaagccagatg (SEQ ID NO:43) (SEQ IDNO:44)  9 gtggcctatgaccgctatgt atggaatgcagatttccagc (SEQ ID NO:45) (SEQID NO:46) 10 ataggcctggtttgtgcatc tgtgggagctacaagtgctg (SEQ ID NO:47)(SEQ ID NO:48) 11 catgatcacactgattgggc ctctgtcgcaaagttcacca (SEQ IDNO:49) (SEQ ID NO:50) 12 gccagagcgcacttacctac ctcaccaaccaggaaatgct (SEQID NO:51) (SEQ ID NO:52) 13a gtgacaattgcagcctctga agtgatattggcgctcgtct1st (SEQ ID NO:53) (SEQ ID NO:54) primer 13b cttcacctctggcaacatcaacttttcccatgaggccttt 2nd (SEQ ID NO:55) (SEQ ID NO:56) primer

[0681] TABLE 3 Predicted Internal Primers for Novel G-Protein CoupledReceptors GPCR GENE INTERNAL OLIGONUCLEOTIDE  1tgtcaccctctgcactatgatgtcatcatggacaggagcacctgtgtccaaagagccactgtgtcttggctgtatggggg (SEQ ID NO:57)  2tccgcctggcttgcaccaacaccaagaagctggaggagactgactttgtcctggcctccctcgtcattgtatcttccttg (SEQ ID NO:58)  3tctgtcctcctggctatgtccgttgactgctatgtggccatctgctgtcccctccattatgcctccatcctcaccaatga (SEQ ID NO:59)  4ttgcttggcccagatgttctttgttcatgggttcacaggtgtggagtctggggtgctcatgctcatggctctagaccgct (SEQ ID NO:60)  5aagagacaaaagatctcttttgctgaccagattctcactgctctggcggtctccagagttggtttgctctgggtattatt (SEQ ID NO:61)  6attctgaacacagccatcaacttcttcctctactgcttcatcagcaagcggttccgcaccatggcagccgccacgctcaag (SEQ ID NO:62)  6bgcgcccatccagaaccgctggctggtacacatcatgtccgacattgccaacatgctagcccttctgaacacagccatcaa (SEQ ID NO:63)  7cctctgaagtcactgaatcccagaaaggctctctacctttagcacaagggaggtcttcaccactggacaaagaaggaacg (SEQ ID NO:64)  8cccacccagtcgtcgtaacagcaacagcaaccctcctctgcccaggtgctaccagtgcaaagctgctaaagtgatcttca (SEQ ID NO:65)  9ggccatctgcaacccactgcagtaccacatcatgatgtccaagaaactctgcattcagatgaccacaggcgccttcata (SEQ ID NO:66) 10ccccagcctgaccatcctttgctcttacatctttattattgccagcatcctccacattcgctccactgagggcaggtcca (SEQ ID NO:67) 11tctcacctgcacacacctatgtactatttcctcagcagtctgtccttcattgacttctgccattccactgtcattacccc (SEQ ID NO:68) 12cagtctgtcatgtggccctcatccacatggtggtccttctcaccatggtgttcttgtctccacagctctttgaatcactg (SEQ ID NO:69) 13atcatggaactctgtctccagtgactttgcattggaacatagactctgatcctgatggtgatctcgccttcacctctggca (SEQ ID NO:70) 13bttgggcagacgagcgccaatatcactgtggagatattgcctgacgaagacccagaactggataaggcattctctgtgtca (SEQ ID NO:71)

[0682]

1 192 1 945 DNA Homo sapiens 1 attactcctg caataatggc aaatctcacaatcgtgactg aatttatcct tatggggttt 60 tctaccaata aaaatatgtg cattttgcattcgattctct tcttgttgat ttatttgtgt 120 gccctgatgg ggaatgtcct cattatcatgatcacaactt tggaccatca tctccacacc 180 cccgtgtatt tcttcttgaa gaatctatctttcttggatc tctgccttat ttcagtcacg 240 gctcccaaat ctatcgccaa ttctttgatacacaacaact ccatttcatt ccttggctgt 300 gtttcccagg tctttttgtt gctttcttcagcatctgcag agctgctcct cctcacggtg 360 atgtcctttg accgctatac tgctatatgtcaccctctgc actatgatgt catcatggac 420 aggagcacct gtgtccaaag agccactgtgtcttggctgt atgggggtct gattgctgtg 480 atgcacacag ctggcacctt ctccttatcctactgtgggt ccaacatggt ccatcagttc 540 ttctgtgaca ttccccagtt attagctatttcttgctcag aaaatttaat aagagaaatt 600 gcactcatcc ttattaatgt agttttggatttctgctgtt ttattgtcat catcattacc 660 tatgtccacg tcttctctac agtcaagaagatcccttcca cagaaggcca gtcaaaagcc 720 tactctattt gccttccaca cttgctggttgtgttatttc tttccactgg attcattgct 780 tatctgaagc cagcttcaga gtctccttctattttggatg ctgtaatttc tgtgttctac 840 actatgctgc ccccaacctt taatcccattatatacagtt tgagaaacaa ggccataaag 900 gtggctctgg ggatgttgat aaagggaaagctcaccaaaa agtaa 945 2 996 DNA Homo sapiens 2 atgagtcctg atgggaaccacagtagtgat ccaacagagt tcgtcctggc agggctccca 60 aatctcaaca gcgcaagagtggaattattt tctgtgtttc ttcttgtcta tctcctgaat 120 ctgacaggca atgtgttgattgtgggggtg gtaagggctg atactcgact acagacccct 180 atgtacttct ttctgggtaacctgtcctgc ctagagatac tgctcacttc tgtcatcatt 240 ccaaagatgc tgagcaatttcctctcaagg caacacacta tttcctttgc tgcatgtatc 300 acccaattct atttctacttctttctcggg gcctccgagt tcttactgtt ggctgtcatg 360 tctgcggatc gctacctggccatctgtcat cctctgcgct accccttgct catgagtggg 420 gctgtgtgct ttcgtgtggccttggcctgc tgggtggggg gactcgtccc tgtgcttggt 480 cccacagtgg ctgtggccttgcttcctttc tgtaagcagg gtgctgtggt acagcacttc 540 ttctgcgaca gtggcccactgctccgcctg gcttgcacca acaccaagaa gctggaggag 600 actgactttg tcctggcctccctcgtcatt gtatcttcct tgctgatcac tgctgtgtcc 660 tacggcctca ttgtgctggcagtcctgagc atcccctctg cttcaggccg tcagaaggcc 720 ttctctacct gtacctcccacttgatagtg gtgaccctct tctatggaag tgccattttt 780 ctctatgtgc ggccatcgcagagtggttct gtggacacta actgggcagt gacagtaata 840 acgacatttg tgacaccactgttgaatcca ttcatctatg ccttacgtaa tgagcaagtc 900 aaggaagctt tgaaggacatgtttaggaag gtagtggcag gcgttttagg gaatctttta 960 cttgataaat gtctcagtgagaaagcagta aagtaa 996 3 954 DNA Homo sapiens 3 atgtcccagg tgactaacaccacacaagaa ggcatctact tcatcctcac ggacatccct 60 ggatttgagg cctcccacatctggatctcc atccccgtct gctgtctcta caccatctcc 120 atcatgggca ataccaccatcctcactgtc attcgcacag agccatctgt ccaccagcgc 180 atgtatctgt ttctctccatgctggccctg acggacctgg gtctcaccct caccacccta 240 cccacagtca tgcagcttctctggttcaac gttcgtagaa tcagctctga ggcctgtttt 300 gctcagtttt tcttccttcatggattctcc tttatggagt cttctgtcct cctggctatg 360 tccgttgact gctatgtggccatctgctgt cccctccatt atgcctccat cctcaccaat 420 gaagtcattg gtagaactgggttagccatc atttgctgct gtgttctggc ggttcttccc 480 tcccttttct tactcaagcgactgcctttc tgccactccc accttctctc tcgctcctat 540 tgcctccacc aggatatgatccgcctggtc tgtgctgaca tcaggctcaa cagctggtat 600 ggatttgctc ttgccttgctcattattatc gtggatcctc tgctcattgt gatctcctat 660 acacttattc tgaaaaatatcttgggcaca gccacctggg ctgagcgact ccgtgccctc 720 aataactgcc tgtcccacattctagctgtc ctggtcctct acattcccat ggttggtgta 780 tctatgactc atcgctttgccaagcatgcc tctccactgg tccatgttat catggccaat 840 atctacctgc tggcacccccggtgatgaac cccatcattt acagtgtaaa gaacaagcag 900 atccaatggg gaatgttaaatttcctttcc ctcaaaaata tgcattcaag atga 954 4 975 DNA Homo sapiens 4atgcctctat ttaattcatt atgctggttt ccaacaattc atgtgactcc tccatctttt 60attcttaatg gaatacctgg tctggaaaga gtacatgtat ggatctccct cccactctgc 120acaatgtaca tcatcttcct tgtggggaat cttggtcttg tgtacctcat ttattatgag 180gagtccttac atcatccgat gtattttttt tttggccatg ctctctccct cattgacctc 240cttacctgca ccaccactct acccaatgca ctctgcatct tctggttcag tctcaaagaa 300attaacttca atgcttgctt ggcccagatg ttctttgttc atgggttcac aggtgtggag 360tctggggtgc tcatgctcat ggctctagac cgctatatag ccatttgcta ccctttgcgt 420tatgctacca cactcaccaa ccctatcatt gccaaggctg agcttgccac cttcctgagg 480ggtgtattgc tgatgattcc tttcccattc ttggttaagc gtttgccttt ctgccaaagc 540aatattatct cccatacgta ctgcgaccac atgtctgtag taaagctatc ttgtgccagc 600atcaaggtca atgtaatcta tggtctaatg gttgctctcc tgattggagt gtttgacatt 660tgttgtatat ctttgtctta cactttgatc ctcaaggcag cgatcagcct ctcttcatca 720gatgctcggc agaaggcttt cagcacctgc actgcccata tatctgccat catcatcacc 780tatgttccag cattcttcac tttctttgcc caccgttttg ggggacacac aattccccct 840tctcttcaca tcattgtggc taatctttat cttcttcttc ccccaactct aaaccctatt 900gtttatggag taaagacaaa acagatacgc aagagtgtca taaagttctt ccagggtgat 960aagggtgcag gttga 975 5 971 DNA Homo sapiens 5 tttttttgtc tgttcagacatgacaacttt tatacccatc attttttcca gtgtggtagt 60 ggttctattt gttattggaaattttgctaa tggcttcata gcattggtaa attccattga 120 gcgggtcaag agacaaaagatctcttttgc tgaccagatt ctcactgctc tggcggtctc 180 cagagttggt ttgctctgggtattattatt aaattggtat tcaactgtgt ttaatccagc 240 tttttatagt gtagaagtaagaactactgc ttataatgtc tgggcagtaa ccggccattt 300 cagcaactgg cttgctactagcctcagcat attttatttg ctcaagattg ccaatttctc 360 caaccttatt tttcttcacttaaagaggag agttaagagt gtcattctgg tgatgctgtt 420 ggggccttta ctatttttggcttgtcaact ttttgtgata aacatgaaag agattgtacg 480 gacaaaagaa tatgaaggaaacttgacttg gaagatcaaa ttgaggagtg cagtgtacct 540 ttcagatgcg actgtaaccacgctaggaaa cttagtgccc ttcactctga ccctgctatg 600 ttttttgctg ttaatctgttctctgtgtaa acatctcaag aagatgcagc tccatggtaa 660 aggatctcaa gatcccagcaccaaggtcca cataaaagct ttgcaaactg tgatcttttt 720 cctcttgtta tgtgccgtttactttctgtc cataatgata tcagtttgga gttttgggag 780 tctggaaaac aaacctgtcttcatgttctg caaagctatt agattcagct atccttcaat 840 ccacccattc atcctgatttggggaaacaa gaagctaaag cagacttttc tttcagtttt 900 gcggcaagtg aggtactgggtgaaaggaga gaagccttca tctccataga ttcatgagag 960 gggcattgtg t 971 6 2189DNA Homo sapiens 6 gggtgtggga tgggagagca tccgcgtgcc tgagtgagcgtgtgagtaca ccgcatgcct 60 gggtattgtg tgtctttgtg tgagcgtggg cgagtgcgtgtttgagtgtc tgcaacatcc 120 ttattcctga ctgaagggcg gcatgcagga ggggcttctcttagccagtc tgtatcctct 180 tcaaacgcac tccagttagg ggacagttca ccggcggccacccccagagt cgccacacac 240 acgcacaggg aggcacgcac ccctctttcc agagccagcaagctcagctt cccttgtcga 300 ggaaaaccga caacgcttta gcatcctcgc tgctccaacagccccacccc ttcccccgtt 360 ctttgcccta ggtatagaat cctctatccg gagatggactggaagcattg gagaggggca 420 agagaggtgg ctgcgaggag agtggagaaa ggcaaactgaggacacgctg gaggagtgag 480 gagccgctgc gggagaggca gatcctaccc acaccacgcccctccctcgg ggttggagat 540 tggggatagc ctgttttcca ctccggtctt ccttcggggctcccaggtct aactgcatct 600 tctcccctga aagtggagcc aagcgaggcg gctgggaccccctcctcttc cgcatccctc 660 ccaccccaca cacactccgc ttccaggcag ccgctgattggctgcgggga gcggcgtccc 720 agccccccgg ctttgaggcg ggagtggagc gggtccgaggtgggaggcgc acagacgggc 780 tccgggagcc cctcccgagg ccccgcgcag cgcgccccgcaccctgcgcc ccgcgccctg 840 cgggagggct gagccaagac tccaggcggg caggtgcggagcgagcagag gggatcacgg 900 ccaagggtag gagccagtcc tgcggggaga gaggcgctgctgctccagct gcctgctgcc 960 tccgcctgcg ccaccaccga gccggcgacc agagtcggggctggcaggcc gggcgcgaag 1020 cggcaagggg agcgaggggc gcgctcatgg agcacacgcacgcccacctc gcagccaaca 1080 gctcgctgtc ttggtggtcc cccggctcgg cctgcggcttgggtttcgtg cccgtggtct 1140 actacagcct cttgctgtgc ctcggtttac cagcaaatatcttgacagtg atcatcctct 1200 cccagctggt ggcaagaaga cagaagtcct cctacaactatctcttggca ctcgctgctg 1260 ccgacatctt ggtcctcttt ttcatagtgt ttgtggacttcctgttggaa gatttcatct 1320 tgaacatgca gatgcctcag gtccccgaca agatcatagaagtgctggaa ttctcatcca 1380 tccacacctc catatggatt actgtaccgt taaccattgacaggtatatc gctgtctgcc 1440 acccgctcaa gtaccacacg gtctcatacc cagcccgcacccggaaagtc attgtaagtg 1500 tttacatcac ctgcttcctg accagcatcc cctattactggtggcccaac atctggactg 1560 aagactacat cagcacctct gtgcatcacg tcctcatctggatccactgc ttcaccgtct 1620 acctggtgcc ctgctccatc ttcttcatct tgaactcaatcattgtgtac aagctcagga 1680 ggaagagcaa ttttcgtctc cgtggctact ccacggggaagaccaccgcc atcttgttca 1740 ccattacctc catctttgcc acactttggg ccccccgcatcatcatgatt ctttaccacc 1800 tctatggggc gcccatccag aaccgctggc tggtacacatcatgtccgac attgccaaca 1860 tgctagccct tctgaacaca gccatcaact tcttcctctactgcttcatc agcaagcggt 1920 tccgcaccat ggcagccgcc acgctcaagg ctttcttcaagtgccagaag caacctgtac 1980 agttctacac caatcataac ttttccataa caagtagcccctggatctcg ccggcaaact 2040 cacactgcat caagatgctg gtgtaccagt atgacaaaaatggaaaaagt cgtaatgaca 2100 gcaaaagctc ctaccagttt gaagatgcca ttggagcttgtgtcatcatc ctgtgaccag 2160 ttaggacaca aagtagagaa gtagtctgt 2189 7 1997DNA Homo sapiens 7 ccagctttca tttcagctcc tgtaggcatc tcctgaattaagcaacacag aaaagtcctc 60 tgaagtcact gaatcccaga aaggctctct acctttagcacaagggaggt cttcaccact 120 ggacaaagaa ggaacgataa ggggtcatca gactggtggtttctgagcat ggattcaacc 180 atcccagtct tgggtacaga actgacacca atcaacggacgtgaggagac tccttgctac 240 aagcagaccc tgagcttcac ggggctgacg tgcatcgtttcccttgtcgc gctgacagga 300 aacgcggttg tgctctggct cctgggctgc cgcatgcgcaggaacgctgt ctccatctac 360 atcctcaacc tggtcgcggc cgacttcctc ttcctctgcttccagattat aaattgcctg 420 gtgtacctca gtaacttctt ctgttccatc tccatcaatttccctagctt cttcaccact 480 gtgatgacct gtgcctacct tgcaggcctg agcatgctgagcaccgtcag caccgagcgc 540 tgcctgtccg tcctgtggcc catctggtat cgctgccgccgccccagaca cctgtcagcg 600 gtcgtgtgtg tcctgctctg ggccctgtcc ctactgctgagcatcttgga agggaagttc 660 tgtggcttct tatttagtga tggtgactct ggttggtgtcagacatttga tttcatcact 720 gcagcgtggc tgattttttt attcatggtt ctctgtgggtccagtctggc cctgctggtc 780 aggatcctct gtggctccag gggtctgcca ctgaccaggctgtacctgac catcctgctc 840 acagtgctgg tgttcctcct ctgcggcctg ccctttggcattcagtggtt cctaatatta 900 tggatctgga aggattctga tgtcttattt tgtcatattcatccagtttc agttgtcctg 960 tcatctctta acagcagtgc caaccccatc atttacttcttcgtgggctc ttttaggaag 1020 cagtggcggc tgcagcagcc gatcctcaag ctggctctccagagggctct gcaggacatt 1080 gctgaggtgg atgaaggtgg agggtggctt cctcaggaaaccctggagct gtcgggaagc 1140 agattggagc agtgaggaag aacctctgcc ctgtcagacaggactttgag agcaatgctg 1200 ccctgccacc cttgacaatt atatgcattt ttcttagccttctgcctcag aaatgtctca 1260 gtggtccctc aaggtcttcg aatagatgtt tatctaacctgacagttgca gttttcaccc 1320 atggaaagca ttagtctgac agtacaatgt ttggattctccttgatatta ccaatacatt 1380 ttccctgtta tcttgcactg aatctttcct actgaacactttttctgcac ttttcattgt 1440 aataaaagga gttgctgtcc acaaccctaa aactcttctttatacttgtt tcctacctga 1500 tagtatcaaa aaggaagatt ccttattaat ctgtcagactatgttcccct gaaaatcatg 1560 ttccctttta tgactggagg cattactgca gttggaagctcaattcttaa taagtgagtt 1620 ctgctacctc taaattccat tgaattctca gatataaagcaaaataatga ccttagagag 1680 agattctccc ttcataaaaa cagtcttaga aattggttttatgaatagcc ctctcctgtc 1740 atttgtccac agcatggtga catgttggcc ttggtttctagtaaagacaa tcgtggcccc 1800 ttccccttga gaactggtaa gttcttattt agctcttcctggactaatga actagtgagg 1860 agcctataaa tatgtcccac cagtttcatt ttggccattggaaacctcaa tattgatttt 1920 aaagtggaaa ttatctttgt ttttccattt attattcacggaaagaaaag aacagaatgt 1980 caaagaaata agaaaaa 1997 8 1458 DNA Homosapiens 8 atgcccatca gcctggccca cggcatcatc cgctcaaccg tgctggttatcttcctcgcc 60 gcctctttcg tcggcaacat agtgctggcg ctagtgttgc agcgcaagccgcagctgctg 120 caggtgacca accgttttat ctttaacctc ctcgtcaccg acctgctgcagatttcgctc 180 gtggccccct gggtggtggc cacctctgtg cctctcttct ggcccctcaacagccacttc 240 tgcacggccc tggttagcct cacccacctg ttcgccttcg ccagcgtcaacaccattgtc 300 gtggtgtcag tggatcgcta cttgtccatc atccaccctc tctcctacccgtccaagatg 360 acccagcgcc gcggttacct gctcctctat ggcacctgga ttgtggccatcctgcagagc 420 actcctccac tctacggctg gggccaggct gcctttgatg agcgcaatgctctctgctcc 480 atgatctggg gggccagccc cagctacact attctcagcg tggtgtccttcatcgtcatt 540 ccactgattg tcatgattgc ctgctactcc gtggtgttct gtgcagcccggaggcagcat 600 gctctgctgt acaatgtcaa gagacacagc ttggaagtgc gagtcaaggactgtgtggag 660 aatgaggatg aagagggagc agagaagaag gaggagttcc aggatgagagtgagtttcgc 720 cgccagcatg aaggtgaggt caaggccaag gagggcagaa tggaagccaaggacggcagc 780 ctgaaggcca aggaaggaag cacggggacc agtgagagta gtgtagaggccaggggcagc 840 gaggaggtca gagagagcag cacggtggcc agcgacggca gcatggagggtaaggaaggc 900 agcaccaaag ttgaggagaa cagcatgaag gcagacaagg gtcgcacagaggtcaaccag 960 tgcagcattg acttgggtga agatgacatg gagtttggtg aagacgacatcaatttcagt 1020 gaggatgacg tcgaggcagt gaacatcccg gagagcctcc cacccagtcgtcgtaacagc 1080 aacagcaacc ctcctctgcc caggtgctac cagtgcaaag ctgctaaagtgatcttcatc 1140 atcattttct cctatgtgct atccctgggg ccctactgct ttttagcagtcctggccgtg 1200 tgggtggatg tcgaaaccca ggtaccccag tgggtgatca ccataatcatctggcttttc 1260 ttcctgcagt gctgcatcca cccctatgtc tatggctaca tgcacaagaccattaagaag 1320 gaaatccagg acatgctgaa gaagttcttc tgcaaggaaa agcccccgaaagaagatagc 1380 cacccagacc tgcccggaac agagggtggg actgaaggca agattgtcccttcctacgat 1440 tctgctactt ttccttga 1458 9 1001 DNA Homo sapiens 9tacaattttt ttcagagagg tcaggaatgg ttgaagaaaa tcataccatg aaaaatgagt 60ttatcctcac aggatttaca gatcaccctg agctgaagac tctgctgttt gtggtgttct 120ttgccatcta tctgatcacc gtggtgggga atattagttt ggtggcactg atatttacac 180actgtcggct tcacacacca atgtacatct ttctgggaaa tctggctctt gtggattctt 240gctgtgcctg tgctattacc cccaaaatgt tagagaactt cttttctgag ggcaaaagga 300tttccctcta tgaatgtgca gtacagtttt attttctttg cactgtggaa actgcagact 360gctttcttct ggcagcagtg gcctatgacc gctatgtggc catctgcaac ccactgcagt 420accacatcat gatgtccaag aaactctgca ttcagatgac cacaggcgcc ttcatagctg 480gaaatctgca ttccatgatt catgtagggc ttgtatttag gttagttttc tgtggattga 540atcacatcaa ccacttttac tgtgatactc ttcccttgta tagactctcc tgtgttgacc 600ctttcatcaa tgaactggtt ctattcatct tctcaggttc agttcaagtc tttaccatag 660gtagtgtctt aatatcttat ctctatattc ttcttactat tttcagaatg aaatccaagg 720agggaagggc caaagccttt tctacttgtg catcccactt ttcatcagtt tcattattct 780atggatctat ttttttccta tacattagac caaatttgct tgaagaagga ggtaatgata 840taccagctgc tattttattt acaatagtag ttcccttact aaatcctttc atttatagtc 900tgagaaacaa ggaagtaata agtgtcttaa gaaaaattct gctgaaaata aaatctcaag 960gaagtgtgaa caaatgacat ctatcctagc ttaatggttt a 1001 10 961 DNA Homosapiens 10 gaaactgact ggaggagatg tcaggagaaa ataattcctc agtgactgagttcattctgg 60 ctgggctctc agaacagcca gagctccagc tgcccctctt cctcctgttcttaggaatct 120 atgtggtcac agtggtgggc aacctgggca tgaccacact gatttggctcagttctcacc 180 tgcacacccc tatgtactat ttcctcagca gtctgtcctt cattgacttctgccattcca 240 ctgtcattac ccctaagatg ctggtgaact ttgtgacaga gaagaacatcatctcctacc 300 ctgaatgcat gactcagctc tacttcttcc tcgtttttgc tattgcagagtgtcacatgt 360 tggctgcaat ggcgtatgac cgttacatgg ccatctgtag ccccttgctgtacagtgtca 420 tcatatccaa taaggcttgc ttttctctga ttttaggggt gtatataataggcctggttt 480 gtgcatcagt tcatacaggc tgtatgttta gggttcaatt ctgcaaatttgatttgatta 540 accattattt ctgtgatctt cttcccctcc taaagctctc ttgctctagtatctatgtca 600 acaaactact tattctatgt gttggtgcat ttaacatcct tgtccccagcctgaccatcc 660 tttgctctta catctttatt attgccagca tcctccacat tcgctccactgagggcaggt 720 ccaaagcctt cagcacttgt agctcccaca tgttggcggt tgtaatcttttttggatctg 780 cagcattcat gtacttgcag ccatcttcaa tcagctccat ggaccaggggaaagtatcct 840 ctgtgtttta tactattatt gtgcccatgt tgaaccctct gatttatagcctgaggaata 900 aagatgtcca tgtttccctg aagaaaatgc tacagagaag aacattattgtaaacagtaa 960 t 961 11 1334 DNA Homo sapiens 11 atgtgtaaat gcttcagaagtggcaatagc actccagtcc tgtgtcaccg aaactcagaa 60 gcatggcagc ccaggaaggccccaagaaca cagcaaactg acatgggtta caccaattta 120 aattccaaga aagagtgcatgtacattaag gaaaatttca aaaagactgt tgacaagatc 180 gtggaccctg gaaaccattcctcagtgact gagtccattc tggctgggct ctcagaacag 240 ccagagctcc agctgcgcctcttcctcctg ttcttaggaa tctgtgtggt cacagtggtg 300 ggcaacttgg gcatgatcacactgattggg ctcagttctc acctgcacac acctatgtac 360 tatttcctca gcagtctgtccttcattgac ttctgccatt ccactgtcat tacccctaag 420 atgctggtga actttgcgacagagaagaac atcatctcct accctgaatg catggctcag 480 ctctatttat tcagtatttttgctattgca gagtgtcaca tgttggctgc aatggcgtat 540 gactgttatg ttgccatctgcagccccttg ctgtacaatg tcatcatgtc ctatcaccac 600 tgcttctggc tcacagtgggagtttacatt ttaggcatcc ttggatctac aattcatacc 660 agttttatgt tgagactctttttgtgcaag actaatgtga ttaaccatta tttttgtgat 720 cttttccctc tcttggggctctcctgctcc agcacctaca tcaatgaatt actggttctg 780 gtcttgagtg catttaacatcctgatgcct gccttaacca tccttgcttc ttacatcttt 840 atcattgcca gcatcctccgcattcactcc actgagggca ggtccaaagc cttcagcact 900 tgcagctccc acatcttggctgttgctgtt ttctttggat ctgcagcatt catgtacctg 960 cagccatcat ctgtcagctccatggaccag aggaaagtgt cgtctgtgtt ttatactact 1020 attgtgccca tgctgaaccccctgatctac agcctgagga ataaagatgt caaacttgcc 1080 gtgaagaaaa ttctgcatcagacagcatgt taatgaatag aatcaatgtt atgttgttac 1140 atcaagatag gtctttggtttgattagata tctaacttat tggatttatt gttgagattt 1200 atgaaaattt agtgatgctcttttatgtaa cacctctcca aaatattcct ccggtctgct 1260 tccatcgaac ttatattccaatgagcatat gtaaagaaat acaaagaata aaatcaaaag 1320 acttttgagg ttta 1334 121015 DNA Homo sapiens 12 tgatatatat agtgtatata tacatacatg cacacatacactatatatat caaggattta 60 tatgatagga ttaattaaga aaaaaattag tggaataaaaataatgttta tgataatttt 120 ggccatagaa tatataatac agatgatgtg aagtacaaaatgttttttat acttcatatt 180 ttgatgtaca aagtatgttt gtctttgtaa ttcagatgattactttgcac ttgtgttccc 240 atgaaaaatg cctttcattt ctaagctggt attggcatctcagccaacac ttttctcctt 300 cttttctgcg tcttctcctt ttctgctttt tctggatctcaggccagagc gcacttacct 360 accagtctgt catgtggccc tcatccacat ggtggtccttctcaccatgg tgttcttgtc 420 tccacagctc tttgaatcac tgaattttca gaatgacttcaaatatgagg catctttcta 480 cctgaggagg gtgatcaggg acctctccat ttgtaccacctgcctcctgg gcatgctgca 540 ggtcgtcaac atcagcccca gcatttcctg gttggtgaggtttaaatgga aatccacaat 600 ttttaccttc catttgttct catggtctct cagttttcctgttagtagta gcctgatctt 660 ttacactgtg gcttcttcca atgtgaccca gatcaatttgcatgtcagta aatactgttc 720 acttttccca ataaactcca taatcagagg actgtttttcactctgtcat tattcagaga 780 tgtttttctt aaacaaataa tgctgttctc aagtgtctacatgatgactc tcattcagga 840 actacaggag atcctggtac cttcacagcc ccagcctctacctaaggatc tttgcagagg 900 caagagccat cagcacatcc tgctgccggt gagtttctcggtgggcatgt acaagatgga 960 cttcatcatc tcaacctcct caacgttgcc atgggcatatgaccgtggtg tctag 1015 13 906 DNA Homo sapiens 13 caaattagaa tgtactcaatgaatggaagg actcttttca aaaagttgtt ccttggcttt 60 ttctttgatt tgtaaattactgatcatcat tttttttttc tatttgttgg aacttgttca 120 tgcagccagt ctaggagtggcttcccaaat tctagtgaca attgcagcct ctgaccacgc 180 tcatggcgta tttgaatttagccctgagtc actctttgtc agtggaactg aaccagaaga 240 tgggtatagc actgttacattaaatgttat aagacatcat ggaactctgt ctccagtgac 300 tttgcattgg aacatagactctgatcctga tggtgatctc gccttcacct ctggcaacat 360 cacatttgag attgggcagacgagcgccaa tatcactgtg gagatattgc ctgacgaaga 420 cccagaactg gataaggcattctctgtgtc agtcctcagt gtttccagtg gttctttggg 480 agctcatatt aatgccacgttaacagtttt ggctagtgat gatccatatg ggatattcat 540 tttttctgag aaaaacagacctgttaaagt tgaggaagca acccagaaca tcacactatc 600 aataataagg ttgaaaggcctcatgggaaa agtccttgtc tcatatgcaa cactagatga 660 tatggaaaaa ccaccttattttccacctaa tttagcgaga gcaactcaag gaagagacta 720 tataccagct tctggatttgctctttttgg agctaatcag agtgaggcaa caatagctat 780 ttcaattttg gatgatgatgagccagaaag gtccgaatct gtctttatcg aactactcaa 840 ctctacttta gtagcgaaagtacagagtcg ttcaagtaag tatcccttag tgtgttatta 900 ttatta 906 14 309 PRTHomo sapiens 14 Met Ala Asn Leu Thr Ile Val Thr Glu Phe Ile Leu Met GlyPhe Ser 1 5 10 15 Thr Asn Lys Asn Met Cys Ile Leu His Ser Ile Leu PheLeu Leu Ile 20 25 30 Tyr Leu Cys Ala Leu Met Gly Asn Val Leu Ile Ile MetIle Thr Thr 35 40 45 Leu Asp His His Leu His Thr Pro Val Tyr Phe Phe LeuLys Asn Leu 50 55 60 Ser Phe Leu Asp Leu Cys Leu Ile Ser Val Thr Ala ProLys Ser Ile 65 70 75 80 Ala Asn Ser Leu Ile His Asn Asn Ser Ile Ser PheLeu Gly Cys Val 85 90 95 Ser Gln Val Phe Leu Leu Leu Ser Ser Ala Ser AlaGlu Leu Leu Leu 100 105 110 Leu Thr Val Met Ser Phe Asp Arg Tyr Thr AlaIle Cys His Pro Leu 115 120 125 His Tyr Asp Val Ile Met Asp Arg Ser ThrCys Val Gln Arg Ala Thr 130 135 140 Val Ser Trp Leu Tyr Gly Gly Leu IleAla Val Met His Thr Ala Gly 145 150 155 160 Thr Phe Ser Leu Ser Tyr CysGly Ser Asn Met Val His Gln Phe Phe 165 170 175 Cys Asp Ile Pro Gln LeuLeu Ala Ile Ser Cys Ser Glu Asn Leu Ile 180 185 190 Arg Glu Ile Ala LeuIle Leu Ile Asn Val Val Leu Asp Phe Cys Cys 195 200 205 Phe Ile Val IleIle Ile Thr Tyr Val His Val Phe Ser Thr Val Lys 210 215 220 Lys Ile ProSer Thr Glu Gly Gln Ser Lys Ala Tyr Ser Ile Cys Leu 225 230 235 240 ProHis Leu Leu Val Val Leu Phe Leu Ser Thr Gly Phe Ile Ala Tyr 245 250 255Leu Lys Pro Ala Ser Glu Ser Pro Ser Ile Leu Asp Ala Val Ile Ser 260 265270 Val Phe Tyr Thr Met Leu Pro Pro Thr Phe Asn Pro Ile Ile Tyr Ser 275280 285 Leu Arg Asn Lys Ala Ile Lys Val Ala Leu Gly Met Leu Ile Lys Gly290 295 300 Lys Leu Thr Lys Lys 305 15 331 PRT Homo sapiens 15 Met SerPro Asp Gly Asn His Ser Ser Asp Pro Thr Glu Phe Val Leu 1 5 10 15 AlaGly Leu Pro Asn Leu Asn Ser Ala Arg Val Glu Leu Phe Ser Val 20 25 30 PheLeu Leu Val Tyr Leu Leu Asn Leu Thr Gly Asn Val Leu Ile Val 35 40 45 GlyVal Val Arg Ala Asp Thr Arg Leu Gln Thr Pro Met Tyr Phe Phe 50 55 60 LeuGly Asn Leu Ser Cys Leu Glu Ile Leu Leu Thr Ser Val Ile Ile 65 70 75 80Pro Lys Met Leu Ser Asn Phe Leu Ser Arg Gln His Thr Ile Ser Phe 85 90 95Ala Ala Cys Ile Thr Gln Phe Tyr Phe Tyr Phe Phe Leu Gly Ala Ser 100 105110 Glu Phe Leu Leu Leu Ala Val Met Ser Ala Asp Arg Tyr Leu Ala Ile 115120 125 Cys His Pro Leu Arg Tyr Pro Leu Leu Met Ser Gly Ala Val Cys Phe130 135 140 Arg Val Ala Leu Ala Cys Trp Val Gly Gly Leu Val Pro Val LeuGly 145 150 155 160 Pro Thr Val Ala Val Ala Leu Leu Pro Phe Cys Lys GlnGly Ala Val 165 170 175 Val Gln His Phe Phe Cys Asp Ser Gly Pro Leu LeuArg Leu Ala Cys 180 185 190 Thr Asn Thr Lys Lys Leu Glu Glu Thr Asp PheVal Leu Ala Ser Leu 195 200 205 Val Ile Val Ser Ser Leu Leu Ile Thr AlaVal Ser Tyr Gly Leu Ile 210 215 220 Val Leu Ala Val Leu Ser Ile Pro SerAla Ser Gly Arg Gln Lys Ala 225 230 235 240 Phe Ser Thr Cys Thr Ser HisLeu Ile Val Val Thr Leu Phe Tyr Gly 245 250 255 Ser Ala Ile Phe Leu TyrVal Arg Pro Ser Gln Ser Gly Ser Val Asp 260 265 270 Thr Asn Trp Ala ValThr Val Ile Thr Thr Phe Val Thr Pro Leu Leu 275 280 285 Asn Pro Phe IleTyr Ala Leu Arg Asn Glu Gln Val Lys Glu Ala Leu 290 295 300 Lys Asp MetPhe Arg Lys Val Val Ala Gly Val Leu Gly Asn Leu Leu 305 310 315 320 LeuAsp Lys Cys Leu Ser Glu Lys Ala Val Lys 325 330 16 317 PRT Homo sapiens16 Met Ser Gln Val Thr Asn Thr Thr Gln Glu Gly Ile Tyr Phe Ile Leu 1 510 15 Thr Asp Ile Pro Gly Phe Glu Ala Ser His Ile Trp Ile Ser Ile Pro 2025 30 Val Cys Cys Leu Tyr Thr Ile Ser Ile Met Gly Asn Thr Thr Ile Leu 3540 45 Thr Val Ile Arg Thr Glu Pro Ser Val His Gln Arg Met Tyr Leu Phe 5055 60 Leu Ser Met Leu Ala Leu Thr Asp Leu Gly Leu Thr Leu Thr Thr Leu 6570 75 80 Pro Thr Val Met Gln Leu Leu Trp Phe Asn Val Arg Arg Ile Ser Ser85 90 95 Glu Ala Cys Phe Ala Gln Phe Phe Phe Leu His Gly Phe Ser Phe Met100 105 110 Glu Ser Ser Val Leu Leu Ala Met Ser Val Asp Cys Tyr Val AlaIle 115 120 125 Cys Cys Pro Leu His Tyr Ala Ser Ile Leu Thr Asn Glu ValIle Gly 130 135 140 Arg Thr Gly Leu Ala Ile Ile Cys Cys Cys Val Leu AlaVal Leu Pro 145 150 155 160 Ser Leu Phe Leu Leu Lys Arg Leu Pro Phe CysHis Ser His Leu Leu 165 170 175 Ser Arg Ser Tyr Cys Leu His Gln Asp MetIle Arg Leu Val Cys Ala 180 185 190 Asp Ile Arg Leu Asn Ser Trp Tyr GlyPhe Ala Leu Ala Leu Leu Ile 195 200 205 Ile Ile Val Asp Pro Leu Leu IleVal Ile Ser Tyr Thr Leu Ile Leu 210 215 220 Lys Asn Ile Leu Gly Thr AlaThr Trp Ala Glu Arg Leu Arg Ala Leu 225 230 235 240 Asn Asn Cys Leu SerHis Ile Leu Ala Val Leu Val Leu Tyr Ile Pro 245 250 255 Met Val Gly ValSer Met Thr His Arg Phe Ala Lys His Ala Ser Pro 260 265 270 Leu Val HisVal Ile Met Ala Asn Ile Tyr Leu Leu Ala Pro Pro Val 275 280 285 Met AsnPro Ile Ile Tyr Ser Val Lys Asn Lys Gln Ile Gln Trp Gly 290 295 300 MetLeu Asn Phe Leu Ser Leu Lys Asn Met His Ser Arg 305 310 315 17 324 PRTHomo sapiens 17 Met Pro Leu Phe Asn Ser Leu Cys Trp Phe Pro Thr Ile HisVal Thr 1 5 10 15 Pro Pro Ser Phe Ile Leu Asn Gly Ile Pro Gly Leu GluArg Val His 20 25 30 Val Trp Ile Ser Leu Pro Leu Cys Thr Met Tyr Ile IlePhe Leu Val 35 40 45 Gly Asn Leu Gly Leu Val Tyr Leu Ile Tyr Tyr Glu GluSer Leu His 50 55 60 His Pro Met Tyr Phe Phe Phe Gly His Ala Leu Ser LeuIle Asp Leu 65 70 75 80 Leu Thr Cys Thr Thr Thr Leu Pro Asn Ala Leu CysIle Phe Trp Phe 85 90 95 Ser Leu Lys Glu Ile Asn Phe Asn Ala Cys Leu AlaGln Met Phe Phe 100 105 110 Val His Gly Phe Thr Gly Val Glu Ser Gly ValLeu Met Leu Met Ala 115 120 125 Leu Asp Arg Tyr Ile Ala Ile Cys Tyr ProLeu Arg Tyr Ala Thr Thr 130 135 140 Leu Thr Asn Pro Ile Ile Ala Lys AlaGlu Leu Ala Thr Phe Leu Arg 145 150 155 160 Gly Val Leu Leu Met Ile ProPhe Pro Phe Leu Val Lys Arg Leu Pro 165 170 175 Phe Cys Gln Ser Asn IleIle Ser His Thr Tyr Cys Asp His Met Ser 180 185 190 Val Val Lys Leu SerCys Ala Ser Ile Lys Val Asn Val Ile Tyr Gly 195 200 205 Leu Met Val AlaLeu Leu Ile Gly Val Phe Asp Ile Cys Cys Ile Ser 210 215 220 Leu Ser TyrThr Leu Ile Leu Lys Ala Ala Ile Ser Leu Ser Ser Ser 225 230 235 240 AspAla Arg Gln Lys Ala Phe Ser Thr Cys Thr Ala His Ile Ser Ala 245 250 255Ile Ile Ile Thr Tyr Val Pro Ala Phe Phe Thr Phe Phe Ala His Arg 260 265270 Phe Gly Gly His Thr Ile Pro Pro Ser Leu His Ile Ile Val Ala Asn 275280 285 Leu Tyr Leu Leu Leu Pro Pro Thr Leu Asn Pro Ile Val Tyr Gly Val290 295 300 Lys Thr Lys Gln Ile Arg Lys Ser Val Ile Lys Phe Phe Gln GlyAsp 305 310 315 320 Lys Gly Ala Gly 18 309 PRT Homo sapiens 18 Met ThrThr Phe Ile Pro Ile Ile Phe Ser Ser Val Val Val Val Leu 1 5 10 15 PheVal Ile Gly Asn Phe Ala Asn Gly Phe Ile Ala Leu Val Asn Ser 20 25 30 IleGlu Arg Val Lys Arg Gln Lys Ile Ser Phe Ala Asp Gln Ile Leu 35 40 45 ThrAla Leu Ala Val Ser Arg Val Gly Leu Leu Trp Val Leu Leu Leu 50 55 60 AsnTrp Tyr Ser Thr Val Phe Asn Pro Ala Phe Tyr Ser Val Glu Val 65 70 75 80Arg Thr Thr Ala Tyr Asn Val Trp Ala Val Thr Gly His Phe Ser Asn 85 90 95Trp Leu Ala Thr Ser Leu Ser Ile Phe Tyr Leu Leu Lys Ile Ala Asn 100 105110 Phe Ser Asn Leu Ile Phe Leu His Leu Lys Arg Arg Val Lys Ser Val 115120 125 Ile Leu Val Met Leu Leu Gly Pro Leu Leu Phe Leu Ala Cys Gln Leu130 135 140 Phe Val Ile Asn Met Lys Glu Ile Val Arg Thr Lys Glu Tyr GluGly 145 150 155 160 Asn Leu Thr Trp Lys Ile Lys Leu Arg Ser Ala Val TyrLeu Ser Asp 165 170 175 Ala Thr Val Thr Thr Leu Gly Asn Leu Val Pro PheThr Leu Thr Leu 180 185 190 Leu Cys Phe Leu Leu Leu Ile Cys Ser Leu CysLys His Leu Lys Lys 195 200 205 Met Gln Leu His Gly Lys Gly Ser Gln AspPro Ser Thr Lys Val His 210 215 220 Ile Lys Ala Leu Gln Thr Val Ile PhePhe Leu Leu Leu Cys Ala Val 225 230 235 240 Tyr Phe Leu Ser Ile Met IleSer Val Trp Ser Phe Gly Ser Leu Glu 245 250 255 Asn Lys Pro Val Phe MetPhe Cys Lys Ala Ile Arg Phe Ser Tyr Pro 260 265 270 Ser Ile His Pro PheIle Leu Ile Trp Gly Asn Lys Lys Leu Lys Gln 275 280 285 Thr Phe Leu SerVal Leu Arg Gln Val Arg Tyr Trp Val Lys Gly Glu 290 295 300 Lys Pro SerSer Pro 305 19 372 PRT Homo sapiens 19 Met Glu His Thr His Ala His LeuAla Ala Asn Ser Ser Leu Ser Trp 1 5 10 15 Trp Ser Pro Gly Ser Ala CysGly Leu Gly Phe Val Pro Val Val Tyr 20 25 30 Tyr Ser Leu Leu Leu Cys LeuGly Leu Pro Ala Asn Ile Leu Thr Val 35 40 45 Ile Ile Leu Ser Gln Leu ValAla Arg Arg Gln Lys Ser Ser Tyr Asn 50 55 60 Tyr Leu Leu Ala Leu Ala AlaAla Asp Ile Leu Val Leu Phe Phe Ile 65 70 75 80 Val Phe Val Asp Phe LeuLeu Glu Asp Phe Ile Leu Asn Met Gln Met 85 90 95 Pro Gln Val Pro Asp LysIle Ile Glu Val Leu Glu Phe Ser Ser Ile 100 105 110 His Thr Ser Ile TrpIle Thr Val Pro Leu Thr Ile Asp Arg Tyr Ile 115 120 125 Ala Val Cys HisPro Leu Lys Tyr His Thr Val Ser Tyr Pro Ala Arg 130 135 140 Thr Arg LysVal Ile Val Ser Val Tyr Ile Thr Cys Phe Leu Thr Ser 145 150 155 160 IlePro Tyr Tyr Trp Trp Pro Asn Ile Trp Thr Glu Asp Tyr Ile Ser 165 170 175Thr Ser Val His His Val Leu Ile Trp Ile His Cys Phe Thr Val Tyr 180 185190 Leu Val Pro Cys Ser Ile Phe Phe Ile Leu Asn Ser Ile Ile Val Tyr 195200 205 Lys Leu Arg Arg Lys Ser Asn Phe Arg Leu Arg Gly Tyr Ser Thr Gly210 215 220 Lys Thr Thr Ala Ile Leu Phe Thr Ile Thr Ser Ile Phe Ala ThrLeu 225 230 235 240 Trp Ala Pro Arg Ile Ile Met Ile Leu Tyr His Leu TyrGly Ala Pro 245 250 255 Ile Gln Asn Arg Trp Leu Val His Ile Met Ser AspIle Ala Asn Met 260 265 270 Leu Ala Leu Leu Asn Thr Ala Ile Asn Phe PheLeu Tyr Cys Phe Ile 275 280 285 Ser Lys Arg Phe Arg Thr Met Ala Ala AlaThr Leu Lys Ala Phe Phe 290 295 300 Lys Cys Gln Lys Gln Pro Val Gln PheTyr Thr Asn His Asn Phe Ser 305 310 315 320 Ile Thr Ser Ser Pro Trp IleSer Pro Ala Asn Ser His Cys Ile Lys 325 330 335 Met Leu Val Tyr Gln TyrAsp Lys Asn Gly Lys Pro Ile Lys Ser Arg 340 345 350 Asn Asp Ser Lys SerSer Tyr Gln Phe Glu Asp Ala Ile Gly Ala Cys 355 360 365 Val Ile Ile Leu370 20 328 PRT Homo sapiens 20 Met Asp Ser Thr Ile Pro Val Leu Gly ThrGlu Leu Thr Pro Ile Asn 1 5 10 15 Gly Arg Glu Glu Thr Pro Cys Tyr LysGln Thr Leu Ser Phe Thr Gly 20 25 30 Leu Thr Cys Ile Val Ser Leu Val AlaLeu Thr Gly Asn Ala Val Val 35 40 45 Leu Trp Leu Leu Gly Cys Arg Met ArgArg Asn Ala Val Ser Ile Tyr 50 55 60 Ile Leu Asn Leu Val Ala Ala Asp PheLeu Phe Leu Cys Phe Gln Ile 65 70 75 80 Ile Asn Cys Leu Val Tyr Leu SerAsn Phe Phe Cys Ser Ile Ser Ile 85 90 95 Asn Phe Pro Ser Phe Phe Thr ThrVal Met Thr Cys Ala Tyr Leu Ala 100 105 110 Gly Leu Ser Met Leu Ser ThrVal Ser Thr Glu Arg Cys Leu Ser Val 115 120 125 Leu Trp Pro Ile Trp TyrArg Cys Arg Arg Pro Arg His Leu Ser Ala 130 135 140 Val Val Cys Val LeuLeu Trp Ala Leu Ser Leu Leu Leu Ser Ile Leu 145 150 155 160 Glu Gly LysPhe Cys Gly Phe Leu Phe Ser Asp Gly Asp Ser Gly Trp 165 170 175 Cys GlnThr Phe Asp Phe Ile Thr Ala Ala Trp Leu Ile Phe Leu Phe 180 185 190 MetVal Leu Cys Gly Ser Ser Leu Ala Leu Leu Val Arg Ile Leu Cys 195 200 205Gly Ser Arg Gly Leu Pro Leu Thr Arg Leu Tyr Leu Thr Ile Leu Leu 210 215220 Thr Val Leu Val Phe Leu Leu Cys Gly Leu Pro Phe Gly Ile Gln Trp 225230 235 240 Phe Leu Ile Leu Trp Ile Trp Lys Asp Ser Asp Val Leu Phe CysHis 245 250 255 Ile His Pro Val Ser Val Val Leu Ser Ser Leu Asn Ser SerAla Asn 260 265 270 Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg Lys GlnTrp Arg Leu 275 280 285 Gln Gln Pro Ile Leu Lys Leu Ala Leu Gln Arg AlaLeu Gln Asp Ile 290 295 300 Ala Glu Val Asp Glu Gly Gly Gly Trp Leu ProGln Glu Thr Leu Glu 305 310 315 320 Leu Ser Gly Ser Arg Leu Glu Gln 32521 485 PRT Homo sapiens 21 Met Pro Ile Ser Leu Ala His Gly Ile Ile ArgSer Thr Val Leu Val 1 5 10 15 Ile Phe Leu Ala Ala Ser Phe Val Gly AsnIle Val Leu Ala Leu Val 20 25 30 Leu Gln Arg Lys Pro Gln Leu Leu Gln ValThr Asn Arg Phe Ile Phe 35 40 45 Asn Leu Leu Val Thr Asp Leu Leu Gln IleSer Leu Val Ala Pro Trp 50 55 60 Val Val Ala Thr Ser Val Pro Leu Phe TrpPro Leu Asn Ser His Phe 65 70 75 80 Cys Thr Ala Leu Val Ser Leu Thr HisLeu Phe Ala Phe Ala Ser Val 85 90 95 Asn Thr Ile Val Val Val Ser Val AspArg Tyr Leu Ser Ile Ile His 100 105 110 Pro Leu Ser Tyr Pro Ser Lys MetThr Gln Arg Arg Gly Tyr Leu Leu 115 120 125 Leu Tyr Gly Thr Trp Ile ValAla Ile Leu Gln Ser Thr Pro Pro Leu 130 135 140 Tyr Gly Trp Gly Gln AlaAla Phe Asp Glu Arg Asn Ala Leu Cys Ser 145 150 155 160 Met Ile Trp GlyAla Ser Pro Ser Tyr Thr Ile Leu Ser Val Val Ser 165 170 175 Phe Ile ValIle Pro Leu Ile Val Met Ile Ala Cys Tyr Ser Val Val 180 185 190 Phe CysAla Ala Arg Arg Gln His Ala Leu Leu Tyr Asn Val Lys Arg 195 200 205 HisSer Leu Glu Val Arg Val Lys Asp Cys Val Glu Asn Glu Asp Glu 210 215 220Glu Gly Ala Glu Lys Lys Glu Glu Phe Gln Asp Glu Ser Glu Phe Arg 225 230235 240 Arg Gln His Glu Gly Glu Val Lys Ala Lys Glu Gly Arg Met Glu Ala245 250 255 Lys Asp Gly Ser Leu Lys Ala Lys Glu Gly Ser Thr Gly Thr SerGlu 260 265 270 Ser Ser Val Glu Ala Arg Gly Ser Glu Glu Val Arg Glu SerSer Thr 275 280 285 Val Ala Ser Asp Gly Ser Met Glu Gly Lys Glu Gly SerThr Lys Val 290 295 300 Glu Glu Asn Ser Met Lys Ala Asp Lys Gly Arg ThrGlu Val Asn Gln 305 310 315 320 Cys Ser Ile Asp Leu Gly Glu Asp Asp MetGlu Phe Gly Glu Asp Asp 325 330 335 Ile Asn Phe Ser Glu Asp Asp Val GluAla Val Asn Ile Pro Glu Ser 340 345 350 Leu Pro Pro Ser Arg Arg Asn SerAsn Ser Asn Pro Pro Leu Pro Arg 355 360 365 Cys Tyr Gln Cys Lys Ala AlaLys Val Ile Phe Ile Ile Ile Phe Ser 370 375 380 Tyr Val Leu Ser Leu GlyPro Tyr Cys Phe Leu Ala Val Leu Ala Val 385 390 395 400 Trp Val Asp ValGlu Thr Gln Val Pro Gln Trp Val Ile Thr Ile Ile 405 410 415 Ile Trp LeuPhe Phe Leu Gln Cys Cys Ile His Pro Tyr Val Tyr Gly 420 425 430 Tyr MetHis Lys Thr Ile Lys Lys Glu Ile Gln Asp Met Leu Lys Lys 435 440 445 PhePhe Cys Lys Glu Lys Pro Pro Lys Glu Asp Ser His Pro Asp Leu 450 455 460Pro Gly Thr Glu Gly Gly Thr Glu Gly Lys Ile Val Pro Ser Tyr Asp 465 470475 480 Ser Ala Thr Phe Pro 485 22 316 PRT Homo sapiens 22 Met Val GluGlu Asn His Thr Met Lys Asn Glu Phe Ile Leu Thr Gly 1 5 10 15 Phe ThrAsp His Pro Glu Leu Lys Thr Leu Leu Phe Val Val Phe Phe 20 25 30 Ala IleTyr Leu Ile Thr Val Val Gly Asn Ile Ser Leu Val Ala Leu 35 40 45 Ile PheThr His Cys Arg Leu His Thr Pro Met Tyr Ile Phe Leu Gly 50 55 60 Asn LeuAla Leu Val Asp Ser Cys Cys Ala Cys Ala Ile Thr Pro Lys 65 70 75 80 MetLeu Glu Asn Phe Phe Ser Glu Gly Lys Arg Ile Ser Leu Tyr Glu 85 90 95 CysAla Val Gln Phe Tyr Phe Leu Cys Thr Val Glu Thr Ala Asp Cys 100 105 110Phe Leu Leu Ala Ala Val Ala Tyr Asp Arg Tyr Val Ala Ile Cys Asn 115 120125 Pro Leu Gln Tyr His Ile Met Met Ser Lys Lys Leu Cys Ile Gln Met 130135 140 Thr Thr Gly Ala Phe Ile Ala Gly Asn Leu His Ser Met Ile His Val145 150 155 160 Gly Leu Val Phe Arg Leu Val Phe Cys Gly Leu Asn His IleAsn His 165 170 175 Phe Tyr Cys Asp Thr Leu Pro Leu Tyr Arg Leu Ser CysVal Asp Pro 180 185 190 Phe Ile Asn Glu Leu Val Leu Phe Ile Phe Ser GlySer Val Gln Val 195 200 205 Phe Thr Ile Gly Ser Val Leu Ile Ser Tyr LeuTyr Ile Leu Leu Thr 210 215 220 Ile Phe Arg Met Lys Ser Lys Glu Gly ArgAla Lys Ala Phe Ser Thr 225 230 235 240 Cys Ala Ser His Phe Ser Ser ValSer Leu Phe Tyr Gly Ser Ile Phe 245 250 255 Phe Leu Tyr Ile Arg Pro AsnLeu Leu Glu Glu Gly Gly Asn Asp Ile 260 265 270 Pro Ala Ala Ile Leu PheThr Ile Val Val Pro Leu Leu Asn Pro Phe 275 280 285 Ile Tyr Ser Leu ArgAsn Lys Glu Val Ile Ser Val Leu Arg Lys Ile 290 295 300 Leu Leu Lys IleLys Ser Gln Gly Ser Val Asn Lys 305 310 315 23 311 PRT Homo sapiens 23Met Ser Gly Glu Asn Asn Ser Ser Val Thr Glu Phe Ile Leu Ala Gly 1 5 1015 Leu Ser Glu Gln Pro Glu Leu Gln Leu Pro Leu Phe Leu Leu Phe Leu 20 2530 Gly Ile Tyr Val Val Thr Val Val Gly Asn Leu Gly Met Thr Thr Leu 35 4045 Ile Trp Leu Ser Ser His Leu His Thr Pro Met Tyr Tyr Phe Leu Ser 50 5560 Ser Leu Ser Phe Ile Asp Phe Cys His Ser Thr Val Ile Thr Pro Lys 65 7075 80 Met Leu Val Asn Phe Val Thr Glu Lys Asn Ile Ile Ser Tyr Pro Glu 8590 95 Cys Met Thr Gln Leu Tyr Phe Phe Leu Val Phe Ala Ile Ala Glu Cys100 105 110 His Met Leu Ala Ala Met Ala Tyr Asp Arg Tyr Met Ala Ile CysSer 115 120 125 Pro Leu Leu Tyr Ser Val Ile Ile Ser Asn Lys Ala Cys PheSer Leu 130 135 140 Ile Leu Gly Val Tyr Ile Ile Gly Leu Val Cys Ala SerVal His Thr 145 150 155 160 Gly Cys Met Phe Arg Val Gln Phe Cys Lys PheAsp Leu Ile Asn His 165 170 175 Tyr Phe Cys Asp Leu Leu Pro Leu Leu LysLeu Ser Cys Ser Ser Ile 180 185 190 Tyr Val Asn Lys Leu Leu Ile Leu CysVal Gly Ala Phe Asn Ile Leu 195 200 205 Val Pro Ser Leu Thr Ile Leu CysSer Tyr Ile Phe Ile Ile Ala Ser 210 215 220 Ile Leu His Ile Arg Ser ThrGlu Gly Arg Ser Lys Ala Phe Ser Thr 225 230 235 240 Cys Ser Ser His MetLeu Ala Val Val Ile Phe Phe Gly Ser Ala Ala 245 250 255 Phe Met Tyr LeuGln Pro Ser Ser Ile Ser Ser Met Asp Gln Gly Lys 260 265 270 Val Ser SerVal Phe Tyr Thr Ile Ile Val Pro Met Leu Asn Pro Leu 275 280 285 Ile TyrSer Leu Arg Asn Lys Asp Val His Val Ser Leu Lys Lys Met 290 295 300 LeuGln Arg Arg Thr Leu Leu 305 310 24 370 PRT Homo sapiens 24 Met Cys LysCys Phe Arg Ser Gly Asn Ser Thr Pro Val Leu Cys His 1 5 10 15 Arg AsnSer Glu Ala Trp Gln Pro Arg Lys Ala Pro Arg Thr Gln Gln 20 25 30 Thr AspMet Gly Tyr Thr Asn Leu Asn Ser Lys Lys Glu Cys Met Tyr 35 40 45 Ile LysGlu Asn Phe Lys Lys Thr Val Asp Lys Ile Val Asp Pro Gly 50 55 60 Asn HisSer Ser Val Thr Glu Ser Ile Leu Ala Gly Leu Ser Glu Gln 65 70 75 80 ProGlu Leu Gln Leu Arg Leu Phe Leu Leu Phe Leu Gly Ile Cys Val 85 90 95 ValThr Val Val Gly Asn Leu Gly Met Ile Thr Leu Ile Gly Leu Ser 100 105 110Ser His Leu His Thr Pro Met Tyr Tyr Phe Leu Ser Ser Leu Ser Phe 115 120125 Ile Asp Phe Cys His Ser Thr Val Ile Thr Pro Lys Met Leu Val Asn 130135 140 Phe Ala Thr Glu Lys Asn Ile Ile Ser Tyr Pro Glu Cys Met Ala Gln145 150 155 160 Leu Tyr Leu Phe Ser Ile Phe Ala Ile Ala Glu Cys His MetLeu Ala 165 170 175 Ala Met Ala Tyr Asp Cys Tyr Val Ala Ile Cys Ser ProLeu Leu Tyr 180 185 190 Asn Val Ile Met Ser Tyr His His Cys Phe Trp LeuThr Val Gly Val 195 200 205 Tyr Ile Leu Gly Ile Leu Gly Ser Thr Ile HisThr Ser Phe Met Leu 210 215 220 Arg Leu Phe Leu Cys Lys Thr Asn Val IleAsn His Tyr Phe Cys Asp 225 230 235 240 Leu Phe Pro Leu Leu Gly Leu SerCys Ser Ser Thr Tyr Ile Asn Glu 245 250 255 Leu Leu Val Leu Val Leu SerAla Phe Asn Ile Leu Met Pro Ala Leu 260 265 270 Thr Ile Leu Ala Ser TyrIle Phe Ile Ile Ala Ser Ile Leu Arg Ile 275 280 285 His Ser Thr Glu GlyArg Ser Lys Ala Phe Ser Thr Cys Ser Ser His 290 295 300 Ile Leu Ala ValAla Val Phe Phe Gly Ser Ala Ala Phe Met Tyr Leu 305 310 315 320 Gln ProSer Ser Val Ser Ser Met Asp Gln Arg Lys Val Ser Ser Val 325 330 335 PheTyr Thr Thr Ile Val Pro Met Leu Asn Pro Leu Ile Tyr Ser Leu 340 345 350Arg Asn Lys Asp Val Lys Leu Ala Val Lys Lys Ile Leu His Gln Thr 355 360365 Ala Cys 370 25 255 PRT Homo sapiens 25 Met Pro Phe Ile Ser Lys LeuVal Leu Ala Ser Gln Pro Thr Leu Phe 1 5 10 15 Ser Phe Phe Ser Ala SerSer Pro Phe Leu Leu Phe Leu Asp Leu Arg 20 25 30 Pro Glu Arg Thr Tyr LeuPro Val Cys His Val Ala Leu Ile His Met 35 40 45 Val Val Leu Leu Thr MetVal Phe Leu Ser Pro Gln Leu Phe Glu Ser 50 55 60 Leu Asn Phe Gln Asn AspPhe Lys Tyr Glu Ala Ser Phe Tyr Leu Arg 65 70 75 80 Arg Val Ile Arg AspLeu Ser Ile Cys Thr Thr Cys Leu Leu Gly Met 85 90 95 Leu Gln Val Val AsnIle Ser Pro Ser Ile Ser Trp Leu Val Arg Phe 100 105 110 Lys Trp Lys SerThr Ile Phe Thr Phe His Leu Phe Ser Trp Ser Leu 115 120 125 Ser Phe ProVal Ser Ser Ser Leu Ile Phe Tyr Thr Val Ala Ser Ser 130 135 140 Asn ValThr Gln Ile Asn Leu His Val Ser Lys Tyr Cys Ser Leu Phe 145 150 155 160Pro Ile Asn Ser Ile Ile Arg Gly Leu Phe Phe Thr Leu Ser Leu Phe 165 170175 Arg Asp Val Phe Leu Lys Gln Ile Met Leu Phe Ser Ser Val Tyr Met 180185 190 Met Thr Leu Ile Gln Glu Leu Gln Glu Ile Leu Val Pro Ser Gln Pro195 200 205 Gln Pro Leu Pro Lys Asp Leu Cys Arg Gly Lys Ser His Gln HisIle 210 215 220 Leu Leu Pro Val Ser Phe Ser Val Gly Met Tyr Lys Met AspPhe Ile 225 230 235 240 Ile Ser Thr Ser Ser Thr Leu Pro Trp Ala Tyr AspArg Gly Val 245 250 255 26 295 PRT Homo sapiens 26 Met Glu Gly Leu PheSer Lys Ser Cys Ser Leu Ala Phe Ser Leu Ile 1 5 10 15 Cys Lys Leu LeuIle Ile Ile Phe Phe Phe Tyr Leu Leu Glu Leu Val 20 25 30 His Ala Ala SerLeu Gly Val Ala Ser Gln Ile Leu Val Thr Ile Ala 35 40 45 Ala Ser Asp HisAla His Gly Val Phe Glu Phe Ser Pro Glu Ser Leu 50 55 60 Phe Val Ser GlyThr Glu Pro Glu Asp Gly Tyr Ser Thr Val Thr Leu 65 70 75 80 Asn Val IleArg His His Gly Thr Leu Ser Pro Val Thr Leu His Trp 85 90 95 Asn Ile AspSer Asp Pro Asp Gly Asp Leu Ala Phe Thr Ser Gly Asn 100 105 110 Ile ThrPhe Glu Ile Gly Gln Thr Ser Ala Asn Ile Thr Val Glu Ile 115 120 125 LeuPro Asp Glu Asp Pro Glu Leu Asp Lys Ala Phe Ser Val Ser Val 130 135 140Leu Ser Val Ser Ser Gly Ser Leu Gly Ala His Ile Asn Ala Thr Leu 145 150155 160 Thr Val Leu Ala Ser Asp Asp Pro Tyr Gly Ile Phe Ile Phe Ser Glu165 170 175 Lys Asn Arg Pro Val Lys Val Glu Glu Ala Thr Gln Asn Ile ThrLeu 180 185 190 Ser Ile Ile Arg Leu Lys Gly Leu Met Gly Lys Val Leu ValSer Tyr 195 200 205 Ala Thr Leu Asp Asp Met Glu Lys Pro Pro Tyr Phe ProPro Asn Leu 210 215 220 Ala Arg Ala Thr Gln Gly Arg Asp Tyr Ile Pro AlaSer Gly Phe Ala 225 230 235 240 Leu Phe Gly Ala Asn Gln Ser Glu Ala ThrIle Ala Ile Ser Ile Leu 245 250 255 Asp Asp Asp Glu Pro Glu Arg Ser GluSer Val Phe Ile Glu Leu Leu 260 265 270 Asn Ser Thr Leu Val Ala Lys ValGln Ser Arg Ser Ser Lys Tyr Pro 275 280 285 Leu Val Cys Tyr Tyr Tyr Asn290 295 27 20 DNA Homo sapiens 27 tcacggctcc caaatctatc 20 28 20 DNAHomo sapiens 28 tgtgcatcac agcaatcaga 20 29 20 DNA Homo sapiens 29tctgtaagca gggtgctgtg 20 30 20 DNA Homo sapiens 30 acaatgaggc cgtaggacac20 31 20 DNA Homo sapiens 31 tctcaccctc accaccctac 20 32 20 DNA Homosapiens 32 cagcagcaaa tgatggctaa 20 33 20 DNA Homo sapiens 33 acctgcaccaccactctacc 20 34 20 DNA Homo sapiens 34 gggaaaggaa tcatcagcaa 20 35 20DNA Homo sapiens 35 taaattccat tgagcgggtc 20 36 20 DNA Homo sapiens 36agcaagccag ttgctgaaat 20 37 20 DNA Homo sapiens 37 gctggtacac atcatgtccg20 38 20 DNA Homo sapiens 38 acaggttgct tctggcactt 20 39 20 DNA Homosapiens 39 cctccatctt tgccacactt 20 40 20 DNA Homo sapiens 40 ggcacttgaagaaagccttg 20 41 20 DNA Homo sapiens 41 cagctcctgt aggcatctcc 20 42 20DNA Homo sapiens 42 caccagtctg atgacccctt 20 43 20 DNA Homo sapiens 43tcagtgagga tgacgtcgag 20 44 20 DNA Homo sapiens 44 gcaggaagaa aagccagatg20 45 20 DNA Homo sapiens 45 gtggcctatg accgctatgt 20 46 20 DNA Homosapiens 46 atggaatgca gatttccagc 20 47 20 DNA Homo sapiens 47 ataggcctggtttgtgcatc 20 48 20 DNA Homo sapiens 48 tgtgggagct acaagtgctg 20 49 20DNA Homo sapiens 49 catgatcaca ctgattgggc 20 50 20 DNA Homo sapiens 50ctctgtcgca aagttcacca 20 51 20 DNA Homo sapiens 51 gccagagcgc acttacctac20 52 20 DNA Homo sapiens 52 ctcaccaacc aggaaatgct 20 53 20 DNA Homosapiens 53 gtgacaattg cagcctctga 20 54 20 DNA Homo sapiens 54 agtgatattggcgctcgtct 20 55 20 DNA Homo sapiens 55 cttcacctct ggcaacatca 20 56 20DNA Homo sapiens 56 acttttccca tgaggccttt 20 57 80 DNA Homo sapiens 57tgtcaccctc tgcactatga tgtcatcatg gacaggagca cctgtgtcca aagagccact 60gtgtcttggc tgtatggggg 80 58 80 DNA Homo sapiens 58 tccgcctggc ttgcaccaacaccaagaagc tggaggagac tgactttgtc ctggcctccc 60 tcgtcattgt atcttccttg 8059 80 DNA Homo sapiens 59 tctgtcctcc tggctatgtc cgttgactgc tatgtggccatctgctgtcc cctccattat 60 gcctccatcc tcaccaatga 80 60 80 DNA Homo sapiens60 ttgcttggcc cagatgttct ttgttcatgg gttcacaggt gtggagtctg gggtgctcat 60gctcatggct ctagaccgct 80 61 80 DNA Homo sapiens 61 aagagacaaa agatctcttttgctgaccag attctcactg ctctggcggt ctccagagtt 60 ggtttgctct gggtattatt 8062 80 DNA Homo sapiens 62 ttctgaacac agccatcaac ttcttcctct actgcttcatcagcaagcgg ttccgcacca 60 tggcagccgc cacgctcaag 80 63 80 DNA Homo sapiens63 gcgcccatcc agaaccgctg gctggtacac atcatgtccg acattgccaa catgctagcc 60cttctgaaca cagccatcaa 80 64 80 DNA Homo sapiens 64 cctctgaagt cactgaatcccagaaaggct ctctaccttt agcacaaggg aggtcttcac 60 cactggacaa agaaggaacg 8065 80 DNA Homo sapiens 65 cccacccagt cgtcgtaaca gcaacagcaa ccctcctctgcccaggtgct accagtgcaa 60 agctgctaaa gtgatcttca 80 66 79 DNA Homo sapiens66 ggccatctgc aacccactgc agtaccacat catgatgtcc aagaaactct gcattcagat 60gaccacaggc gccttcata 79 67 80 DNA Homo sapiens 67 ccccagcctg accatcctttgctcttacat ctttattatt gccagcatcc tccacattcg 60 ctccactgag ggcaggtcca 8068 80 DNA Homo sapiens 68 tctcacctgc acacacctat gtactatttc ctcagcagtctgtccttcat tgacttctgc 60 cattccactg tcattacccc 80 69 80 DNA Homo sapiens69 cagtctgtca tgtggccctc atccacatgg tggtccttct caccatggtg ttcttgtctc 60cacagctctt tgaatcactg 80 70 80 DNA Homo sapiens 70 tcatggaact ctgtctccagtgactttgca ttggaacata gactctgatc ctgatggtga 60 tctcgccttc acctctggca 8071 80 DNA Homo sapiens 71 ttgggcagac gagcgccaat atcactgtgg agatattgcctgacgaagac ccagaactgg 60 ataaggcatt ctctgtgtca 80 72 321 PRT Homosapiens 72 Met Val Asn Leu Thr Ser Met Ser Gly Phe Leu Leu Met Gly PheSer 1 5 10 15 Asp Glu Arg Lys Leu Gln Ile Leu His Ala Leu Val Phe LeuVal Thr 20 25 30 Tyr Leu Leu Ala Leu Thr Gly Asn Leu Leu Ile Ile Thr IleIle Thr 35 40 45 Val Asp Arg Arg Leu His Ser Pro Met Tyr Tyr Phe Leu LysHis Leu 50 55 60 Ser Leu Leu Asp Leu Cys Phe Ile Ser Val Thr Val Pro GlnSer Ile 65 70 75 80 Ala Asn Ser Leu Met Gly Asn Gly Tyr Ile Ser Leu ValGln Cys Ile 85 90 95 Leu Gln Val Phe Phe Phe Ile Ala Leu Ala Ser Ser GluVal Ala Ile 100 105 110 Leu Thr Val Met Ser Tyr Asp Arg Tyr Ala Ala IleCys Gln Pro Leu 115 120 125 His Tyr Glu Thr Ile Met Asp Pro Arg Ala CysArg His Ala Val Ile 130 135 140 Ala Val Trp Ile Ala Gly Gly Leu Ser GlyLeu Met His Ala Ala Ile 145 150 155 160 Asn Phe Ser Ile Pro Leu Cys GlyLys Arg Val Ile His Gln Phe Phe 165 170 175 Cys Asp Val Pro Gln Met LeuLys Leu Ala Cys Ser Tyr Glu Phe Ile 180 185 190 Asn Glu Ile Ala Leu AlaAla Phe Thr Thr Ser Ala Ala Phe Ile Cys 195 200 205 Leu Ile Ser Ile ValLeu Ser Tyr Ile Arg Ile Phe Ser Thr Val Leu 210 215 220 Arg Ile Pro SerAla Glu Gly Arg Thr Lys Val Phe Ser Thr Cys Leu 225 230 235 240 Pro HisLeu Phe Val Ala Thr Phe Phe Leu Ser Ala Ala Gly Phe Glu 245 250 255 PheLeu Arg Leu Pro Ser Asp Ser Ser Ser Thr Val Asp Leu Val Phe 260 265 270Ser Val Phe Tyr Thr Val Ile Pro Pro Thr Leu Asn Pro Val Ile Tyr 275 280285 Ser Leu Arg Asn Asp Ser Met Lys Ala Ala Leu Arg Lys Met Leu Ser 290295 300 Lys Glu Glu Leu Pro Gln Arg Lys Met Cys Leu Lys Ala Met Phe Lys305 310 315 320 Leu 73 417 PRT Homo sapiens 73 Met Glu Ser Ser Pro IlePro Gln Ser Ser Gly Asn Ser Ser Thr Leu 1 5 10 15 Gly Arg Val Pro GlnThr Pro Gly Pro Ser Thr Ala Ser Gly Val Pro 20 25 30 Glu Val Gly Leu ArgAsp Val Ala Ser Glu Ser Val Ala Leu Phe Phe 35 40 45 Met Leu Leu Leu AspLeu Thr Ala Val Ala Gly Asn Ala Ala Val Met 50 55 60 Ala Val Ile Ala LysThr Pro Ala Leu Arg Lys Phe Val Phe Val Phe 65 70 75 80 His Leu Cys LeuVal Asp Leu Leu Ala Ala Leu Thr Leu Met Pro Leu 85 90 95 Ala Met Leu SerSer Pro Ala Leu Phe Asp His Ala Leu Phe Gly Glu 100 105 110 Val Ala CysArg Leu Tyr Leu Phe Leu Ser Val Cys Phe Val Ser Leu 115 120 125 Ala IleLeu Ser Val Ser Ala Ile Asn Val Glu Arg Tyr Tyr Tyr Val 130 135 140 ValHis Pro Met Arg Tyr Glu Val Arg Met Thr Leu Gly Leu Val Ala 145 150 155160 Ser Val Leu Val Gly Val Trp Val Lys Ala Leu Ala Met Ala Ser Val 165170 175 Pro Val Leu Gly Arg Val Ser Trp Glu Glu Gly Ala Pro Ser Val Pro180 185 190 Pro His Cys Ser Leu Gln Trp Ser His Ser Ala Tyr Cys Gln LeuPhe 195 200 205 Val Val Val Phe Ala Val Leu Tyr Phe Leu Leu Pro Leu LeuLeu Ile 210 215 220 Leu Leu Val Tyr Cys Ser Met Phe Arg Val Ala Arg ValAla Ala Met 225 230 235 240 Pro Asp Gly Pro Leu Pro Thr Trp Met Glu ThrPro Arg Gln Arg Ser 245 250 255 Glu Ser Leu Ser Ser Arg Ser Thr Met ValThr Ser Ser Gly Ala Pro 260 265 270 Gln Thr Thr Pro His Arg Thr Phe GlyGly Gly Lys Ala Ala Val Val 275 280 285 Leu Leu Ala Val Gly Gly Gln PheLeu Leu Cys Trp Leu Pro Tyr Phe 290 295 300 Ser Phe His Leu Tyr Val AlaLeu Ser Ala Gln Pro Ile Ser Thr Gly 305 310 315 320 Gln Val Glu Ser ValVal Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser 325 330 335 Asn Pro Phe PheTyr Gly Cys Leu Asn Arg Gln Ile Arg Gly Glu Leu 340 345 350 Ser Lys GlnPhe Val Cys Phe Phe Lys Pro Ala Pro Glu Glu Glu Leu 355 360 365 Arg LeuPro Ser Arg Glu Gly Ser Ile Glu Glu Asn Phe Leu Gln Phe 370 375 380 LeuGln Gly Thr Gly Cys Pro Ser Glu Ser Trp Val Ser Arg Pro Leu 385 390 395400 Pro Ser Pro Lys Gln Glu Pro Pro Ala Val Asp Phe Arg Ile Gln Ala 405410 415 Arg 74 252 PRT Homo sapiens 74 Val Asp Leu Leu Ala Ala Leu ThrLeu Met Pro Leu Ala Met Leu Ser 1 5 10 15 Ser Ser Ala Leu Phe Asp HisAla Leu Phe Gly Glu Val Ala Cys Arg 20 25 30 Leu Tyr Leu Phe Leu Ser ValCys Phe Val Ser Leu Ala Ile Leu Ser 35 40 45 Val Ser Ala Ile Asn Val GluArg Tyr Tyr Tyr Val Val His Pro Met 50 55 60 Arg Tyr Glu Val Arg Met LysLeu Gly Leu Val Ala Ser Val Leu Val 65 70 75 80 Gly Val Trp Val Lys AlaLeu Ala Met Ala Ser Val Pro Val Leu Gly 85 90 95 Arg Val Ser Trp Glu GluGly Pro Pro Ser Val Pro Pro Gly Cys Ser 100 105 110 Leu Gln Trp Ser HisSer Ala Tyr Cys Gln Leu Phe Val Val Val Phe 115 120 125 Ala Val Leu TyrPhe Leu Leu Pro Leu Leu Leu Ile Leu Val Val Tyr 130 135 140 Cys Ser MetPhe Arg Val Ala Arg Val Ala Ala Met Gln His Gly Pro 145 150 155 160 LeuPro Thr Trp Met Glu Thr Pro Arg Gln Arg Ser Glu Ser Leu Ser 165 170 175Ser Arg Ser Thr Met Val Thr Ser Ser Gly Ala Pro Gln Thr Thr Pro 180 185190 His Arg Thr Phe Gly Gly Gly Lys Ala Ala Val Val Leu Leu Ala Val 195200 205 Gly Gly Gln Phe Leu Leu Cys Trp Leu Pro Tyr Phe Ser Phe His Leu210 215 220 Tyr Val Ala Leu Ser Ala Gln Pro Ile Ala Ala Gly Gln Val GluAsn 225 230 235 240 Val Val Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser 245250 75 319 PRT Mus musculus 75 Met Ala Thr Ser Asn Ser Ser Thr Ile ValSer Ser Thr Phe Tyr Leu 1 5 10 15 Thr Gly Ile Pro Gly Tyr Glu Glu PheHis His Trp Ile Ser Ile Pro 20 25 30 Phe Cys Phe Leu Tyr Leu Val Gly IleThr Gly Asn Cys Met Ile Leu 35 40 45 His Ile Val Arg Thr Asp Pro Arg LeuHis Glu Pro Met Tyr Tyr Phe 50 55 60 Leu Ala Met Leu Ser Leu Thr Asp MetAla Met Ser Leu Pro Thr Met 65 70 75 80 Met Ser Leu Phe Arg Val Leu TrpSer Ile Ser Arg Glu Ile Gln Phe 85 90 95 Asn Ile Cys Val Val Gln Met PheLeu Ile His Thr Phe Ser Phe Thr 100 105 110 Glu Ser Ser Val Leu Leu AlaMet Ala Leu Asp Arg Tyr Val Ala Ile 115 120 125 Cys His Pro Leu Arg TyrAla Thr Ile Leu Thr Pro Lys Leu Ile Ala 130 135 140 Lys Ile Gly Thr AlaAla Leu Leu Arg Ser Ser Ile Leu Ile Ile Pro 145 150 155 160 Leu Ile AlaArg Leu Ala Phe Phe Pro Phe Cys Gly Ser His Val Leu 165 170 175 Ser HisSer Tyr Cys Leu His Gln Asp Met Ile Arg Leu Ala Cys Ala 180 185 190 AspIle Arg Phe Asn Val Ile Tyr Gly Leu Val Leu Ile Thr Leu Leu 195 200 205Trp Gly Met Asp Ser Leu Gly Ile Phe Val Ser Tyr Val Leu Ile Leu 210 215220 His Ser Val Leu Lys Ile Ala Ser Arg Glu Gly Arg Leu Lys Ala Leu 225230 235 240 Asn Thr Cys Ala Ser His Ile Cys Ala Val Leu Ile Leu Tyr ValPro 245 250 255 Met Ile Gly Leu Ser Ile Val His Arg Phe Ala Lys His SerSer Pro 260 265 270 Leu Ile His Ile Phe Met Ala His Ile Tyr Leu Leu ValPro Pro Val 275 280 285 Leu Asn Pro Ile Ile Tyr Ser Val Lys Thr Lys GlnIle Arg Glu Gly 290 295 300 Ile Leu His Leu Leu Cys Ser Pro Lys Ile SerSer Ile Thr Met 305 310 315 76 326 PRT Mus musculus 76 Met Lys Val AlaSer Ser Phe His Asn Asp Thr Asn Pro Gln Asp Val 1 5 10 15 Trp Tyr ValLeu Ile Gly Ile Pro Gly Leu Glu Asp Leu His Ser Trp 20 25 30 Ile Ala IlePro Ile Cys Ser Met Tyr Ile Val Ala Val Ile Gly Asn 35 40 45 Val Leu LeuIle Phe Leu Ile Val Thr Glu Arg Ser Leu His Glu Pro 50 55 60 Met Tyr PhePhe Leu Ser Met Leu Ala Leu Ala Asp Leu Leu Leu Ser 65 70 75 80 Thr AlaThr Ala Pro Lys Met Leu Ala Ile Phe Trp Phe His Ser Arg 85 90 95 Gly IleSer Phe Gly Ser Cys Val Ser Gln Met Phe Phe Ile His Phe 100 105 110 IlePhe Val Ala Glu Ser Ala Ile Leu Leu Ala Met Ala Phe Asp Arg 115 120 125Tyr Val Ala Ile Cys Tyr Pro Leu Arg Tyr Thr Thr Ile Leu Thr Ser 130 135140 Ser Val Ile Gly Lys Ile Gly Thr Ala Ala Val Val Arg Ser Phe Leu 145150 155 160 Ile Cys Phe Pro Phe Ile Phe Leu Val Tyr Arg Leu Leu Tyr CysGly 165 170 175 Lys His Ile Ile Pro His Ser Tyr Cys Glu His Met Gly IleAla Arg 180 185 190 Leu Ala Cys Asp Asn Ile Thr Val Asn Ile Ile Tyr GlyLeu Thr Met 195 200 205 Ala Leu Leu Ser Thr Gly Leu Asp Ile Leu Leu IleIle Ile Ser Tyr 210 215 220 Thr Met Ile Leu Arg Thr Val Phe Gln Ile ProSer Trp Ala Ala Arg 225 230 235 240 Tyr Lys Ala Leu Asn Thr Cys Gly SerHis Ile Cys Val Ile Leu Leu 245 250 255 Phe Tyr Thr Pro Ala Phe Phe SerPhe Phe Ala His Arg Phe Gly Gly 260 265 270 Lys Thr Val Pro Arg His IleHis Ile Leu Val Ala Asn Leu Tyr Val 275 280 285 Val Val Pro Pro Met LeuAsn Pro Ile Ile Tyr Gly Val Lys Thr Lys 290 295 300 Gln Ile Gln Asp ArgVal Val Phe Leu Phe Ser Ser Val Ser Thr Cys 305 310 315 320 Gln His AspSer Arg Cys 325 77 303 PRT Homo sapiens 77 Met Glu Ser Ala Leu Pro SerIle Phe Thr Leu Val Ile Ile Ala Glu 1 5 10 15 Phe Ile Ile Gly Asn LeuSer Asn Gly Phe Ile Val Leu Ile Asn Cys 20 25 30 Ile Asp Trp Val Ser LysArg Glu Leu Ser Ser Val Asp Lys Leu Leu 35 40 45 Ile Ile Leu Ala Ile SerArg Ile Gly Leu Ile Trp Glu Ile Leu Val 50 55 60 Ser Trp Phe Leu Ala LeuHis Tyr Leu Ala Ile Phe Val Ser Gly Thr 65 70 75 80 Gly Leu Arg Ile MetIle Phe Ser Trp Ile Val Ser Asn His Phe Asn 85 90 95 Leu Trp Leu Ala ThrIle Phe Ser Ile Phe Tyr Leu Leu Lys Ile Ala 100 105 110 Ser Phe Ser SerPro Ala Phe Leu Tyr Leu Lys Trp Arg Val Asn Lys 115 120 125 Val Ile LeuMet Ile Leu Leu Gly Thr Leu Val Phe Leu Phe Leu Asn 130 135 140 Leu IleGln Ile Asn Met His Ile Lys Asp Trp Leu Asp Arg Tyr Glu 145 150 155 160Arg Asn Thr Thr Trp Asn Phe Ser Met Ser Asp Phe Glu Thr Phe Ser 165 170175 Val Ser Val Lys Phe Thr Met Thr Met Phe Ser Leu Thr Pro Phe Thr 180185 190 Val Ala Phe Ile Ser Phe Leu Leu Leu Ile Phe Ser Leu Gln Lys His195 200 205 Leu Gln Lys Met Gln Leu Asn Tyr Lys Gly His Arg Asp Pro ArgThr 210 215 220 Lys Val His Thr Asn Ala Leu Lys Ile Val Ile Ser Phe LeuLeu Phe 225 230 235 240 Tyr Ala Ser Phe Phe Leu Cys Val Leu Ile Ser TrpIle Ser Glu Leu 245 250 255 Tyr Gln Asn Thr Val Ile Tyr Met Leu Cys GluThr Ile Gly Val Phe 260 265 270 Ser Pro Ser Ser His Ser Phe Leu Leu IleLeu Gly Asn Ala Lys Leu 275 280 285 Arg Gln Ala Phe Leu Leu Val Ala AlaLys Val Trp Ala Lys Arg 290 295 300 78 375 PRT Homo sapiens 78 Glu SerPro Arg Asn Lys Asp Phe Thr Asp Lys Val Pro Trp Asn Gln 1 5 10 15 ArgGlu Ala Gly Met Glu Thr Pro Asn Thr Thr Glu Asp Tyr Asp Thr 20 25 30 ThrThr Glu Phe Asp Tyr Gly Asp Ala Thr Pro Cys Gln Lys Val Asn 35 40 45 GluArg Ala Phe Gly Ala Gln Leu Leu Pro Pro Leu Tyr Ser Leu Val 50 55 60 PheVal Ile Gly Leu Val Gly Asn Ile Leu Val Val Leu Val Leu Val 65 70 75 80Gln Tyr Lys Arg Leu Lys Asn Met Thr Ser Ile Tyr Leu Leu Asn Leu 85 90 95Ala Ile Ser Asp Leu Leu Phe Leu Phe Thr Leu Pro Phe Trp Ile Asp 100 105110 Tyr Lys Leu Lys Asp Asp Trp Val Phe Gly Asp Ala Met Cys Lys Ile 115120 125 Leu Ser Gly Phe Tyr Tyr Thr Gly Leu Tyr Ser Glu Ile Phe Phe Ile130 135 140 Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val His Ala ValPhe 145 150 155 160 Ala Leu Arg Ala Arg Thr Val Thr Phe Gly Val Ile ThrSer Ile Ile 165 170 175 Ile Trp Ala Leu Ala Ile Leu Ala Ser Met Pro GlyLeu Tyr Phe Ser 180 185 190 Lys Thr Gln Trp Glu Phe Thr His His Thr CysSer Leu His Phe Pro 195 200 205 His Glu Ser Leu Arg Glu Trp Lys Leu PheGln Ala Leu Lys Leu Asn 210 215 220 Leu Phe Gly Leu Val Leu Pro Leu LeuVal Met Ile Ile Cys Tyr Thr 225 230 235 240 Gly Ile Ile Lys Ile Leu LeuArg Arg Pro Asn Glu Lys Lys Ser Lys 245 250 255 Ala Val Arg Leu Ile PheVal Ile Met Ile Ile Phe Phe Leu Phe Trp 260 265 270 Thr Pro Tyr Asn LeuThr Ile Leu Ile Ser Val Phe Gln Asp Phe Leu 275 280 285 Phe Thr His GluCys Glu Gln Ser Arg His Leu Asp Leu Ala Val Gln 290 295 300 Val Thr GluVal Ile Ala Tyr Thr His Cys Cys Val Asn Pro Val Ile 305 310 315 320 TyrAla Phe Val Gly Glu Arg Phe Arg Lys Tyr Leu Arg Gln Leu Phe 325 330 335His Arg Arg Val Ala Val His Leu Val Lys Trp Leu Pro Phe Leu Ser 340 345350 Val Asp Arg Leu Glu Arg Val Ser Ser Thr Ser Pro Ser Thr Gly Glu 355360 365 His Glu Leu Ser Ala Gly Phe 370 375 79 314 PRT Homo sapiens 79Met Asp Ser Thr Ile Pro Val Leu Gly Thr Glu Leu Thr Pro Ile Asn 1 5 1015 Gly Arg Glu Glu Thr Pro Cys Tyr Lys Gln Thr Leu Ser Phe Thr Gly 20 2530 Leu Thr Cys Ile Val Ser Leu Val Ala Leu Thr Gly Asn Ala Val Val 35 4045 Leu Trp Leu Leu Gly Cys Arg Met Arg Arg Asn Ala Val Ser Ile Tyr 50 5560 Ile Leu Asn Leu Val Ala Ala Asp Phe Leu Phe Leu Ser Gly His Ile 65 7075 80 Ile Cys Ser Pro Leu Arg Leu Ile Asn Ile Arg His Pro Ile Ser Lys 8590 95 Ile Leu Ser Pro Val Met Thr Phe Pro Tyr Phe Ile Gly Leu Ser Met100 105 110 Leu Ser Ala Ile Ser Thr Glu Arg Cys Leu Ser Ile Leu Trp ProIle 115 120 125 Trp Tyr His Cys Arg Arg Pro Arg Tyr Leu Ser Ser Val MetCys Val 130 135 140 Leu Leu Trp Ala Leu Ser Leu Leu Arg Ser Ile Leu GluTrp Met Phe 145 150 155 160 Cys Asp Phe Leu Phe Ser Gly Ala Asp Ser ValTrp Cys Glu Thr Ser 165 170 175 Asp Phe Ile Thr Ile Ala Trp Leu Val PheLeu Cys Val Val Leu Cys 180 185 190 Gly Ser Ser Leu Val Leu Leu Val ArgIle Leu Cys Gly Ser Arg Lys 195 200 205 Met Pro Leu Thr Arg Leu Tyr ValThr Ile Leu Leu Thr Val Leu Val 210 215 220 Phe Leu Leu Cys Gly Leu ProPhe Gly Ile Gln Trp Ala Leu Phe Ser 225 230 235 240 Arg Ile His Leu AspTrp Lys Val Leu Phe Cys His Val His Leu Val 245 250 255 Ser Ile Phe LeuSer Ala Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr 260 265 270 Phe Phe ValGly Ser Phe Arg Gln Arg Gln Asn Arg Gln Asn Leu Gln 275 280 285 Asp ThrPro Glu Val Asp Glu Gly Gly Gly Trp Leu Pro Gln Glu Thr 290 295 300 LeuGlu Leu Ser Gly Ser Arg Leu Glu Gln 305 310 80 407 PRT Homo sapiens 80Met Ser Leu Asn Ser Ser Leu Ser Cys Arg Lys Glu Leu Ser Asn Leu 1 5 1015 Thr Glu Glu Glu Gly Gly Glu Gly Gly Val Ile Ile Thr Gln Phe Ile 20 2530 Ala Ile Ile Val Ile Thr Ile Phe Val Cys Leu Gly Asn Leu Val Ile 35 4045 Val Val Thr Leu Tyr Lys Lys Ser Tyr Leu Leu Thr Leu Ser Asn Lys 50 5560 Phe Val Phe Ser Leu Thr Leu Ser Asn Phe Leu Leu Ser Val Leu Val 65 7075 80 Leu Pro Phe Val Val Thr Ser Ser Ile Arg Arg Glu Trp Ile Phe Gly 8590 95 Val Val Trp Cys Asn Phe Ser Ala Leu Leu Tyr Leu Leu Ile Ser Ser100 105 110 Ala Ser Met Leu Thr Leu Gly Val Ile Ala Ile Asp Arg Tyr TyrAla 115 120 125 Val Leu Tyr Pro Met Val Tyr Pro Met Lys Ile Thr Gly AsnArg Ala 130 135 140 Val Met Ala Leu Val Tyr Ile Trp Leu His Ser Leu IleGly Cys Leu 145 150 155 160 Pro Pro Leu Phe Gly Trp Ser Ser Val Glu PheAsp Glu Phe Lys Trp 165 170 175 Met Cys Val Ala Ala Trp His Arg Glu ProGly Tyr Thr Ala Phe Trp 180 185 190 Gln Ile Trp Cys Ala Leu Phe Pro PheLeu Val Met Leu Val Cys Tyr 195 200 205 Gly Phe Ile Phe Arg Val Ala ArgVal Lys Ala Arg Lys Val His Cys 210 215 220 Gly Thr Val Val Ile Val GluGlu Asp Ala Gln Arg Thr Gly Arg Lys 225 230 235 240 Asn Ser Ser Thr SerThr Ser Ser Ser Gly Ser Arg Arg Asn Ala Phe 245 250 255 Gln Gly Val ValTyr Ser Ala Asn Gln Cys Lys Ala Leu Ile Thr Ile 260 265 270 Leu Val ValLeu Gly Ala Phe Met Val Thr Trp Gly Pro Tyr Met Val 275 280 285 Val IleAla Ser Glu Ala Leu Trp Gly Lys Ser Ser Val Ser Pro Ser 290 295 300 LeuGlu Thr Trp Ala Thr Trp Leu Ser Phe Ala Ser Ala Val Cys His 305 310 315320 Pro Leu Ile Tyr Gly Leu Trp Asn Lys Thr Val Arg Lys Glu Leu Leu 325330 335 Gly Met Cys Phe Gly Asp Arg Tyr Tyr Arg Glu Pro Phe Val Gln Arg340 345 350 Gln Arg Thr Ser Arg Leu Phe Ser Ile Ser Asn Arg Ile Thr AspLeu 355 360 365 Gly Leu Ser Pro His Leu Thr Ala Leu Met Ala Gly Gly GlnPro Leu 370 375 380 Gly His Ser Ser Ser Thr Gly Asp Thr Gly Phe Ser CysSer Gln Asp 385 390 395 400 Ser Gly Asn Leu Arg Ala Leu 405 81 313 PRTHylobates lar 81 Met Ala Asn Glu Asn Tyr Thr Lys Val Thr Glu Phe Ile PheThr Gly 1 5 10 15 Leu Asn Tyr Asn Pro Gln Leu Gln Val Phe Leu Phe LeuLeu Phe Leu 20 25 30 Thr Phe Tyr Val Ile Ser Val Thr Gly Asn Phe Gly MetIle Val Leu 35 40 45 Ile Arg Met Asp Ser Arg Leu His Thr Pro Met Tyr PhePhe Leu Ser 50 55 60 His Leu Ser Phe Val Asp Ile Cys Phe Ser Ser Val ValSer Pro Lys 65 70 75 80 Met Leu Thr Asp Phe Phe Val Lys Arg Lys Ala IleSer Phe Leu Gly 85 90 95 Cys Ala Leu Gln Gln Trp Phe Phe Gly Phe Phe ValAla Ala Glu Cys 100 105 110 Phe Leu Leu Ala Ser Met Ala Tyr Asp Arg TyrVal Ala Ile Cys Asn 115 120 125 Pro Leu Leu Tyr Ser Val Phe Met Ser GlnArg Leu Cys Ile Gln Leu 130 135 140 Val Val Gly Pro Tyr Val Ile Gly LeuMet Asn Thr Met Thr His Thr 145 150 155 160 Thr Asn Ala Phe Arg Leu ProPhe Cys Gly Leu Asn Val Ile Asn His 165 170 175 Phe Phe Cys Asp Met SerPro Leu Leu Ser Leu Val Cys Ala Asp Thr 180 185 190 Arg Leu Asn Lys LeuAla Val Phe Ile Met Ala Gly Ala Val Gly Val 195 200 205 Phe Ser Gly LeuThr Ile Leu Ile Ser Tyr Ile Tyr Ile Leu Met Ala 210 215 220 Ile Leu ArgIle Arg Ser Ala Asp Gly Arg Cys Lys Thr Phe Ser Thr 225 230 235 240 CysSer Ser His Leu Thr Ala Val Phe Ile Leu Tyr Gly Thr Leu Phe 245 250 255Phe Ile Tyr Val Arg Pro Ser Ala Ser Phe Pro Leu Asp Leu Asn Lys 260 265270 Val Val Ser Val Phe Tyr Thr Ala Val Ile Pro Met Leu Asn Pro Leu 275280 285 Ile Tyr Ser Leu Arg Asn Lys Glu Val Lys Asp Ala Ile His Arg Thr290 295 300 Val Thr Gln Arg Lys Phe Cys Lys Ala 305 310 82 314 PRT Musmusculus 82 Met Asn Asp Met Thr Ser Gly Asn Tyr Cys Thr Val Thr Glu PhePhe 1 5 10 15 Leu Ala Gly Leu Ser Glu Lys Pro Glu Leu Gln Leu Pro LeuPhe Phe 20 25 30 Leu Phe Ile Gly Ile Tyr Met Ile Thr Val Ala Gly Asn LeuGly Met 35 40 45 Ile Ile Leu Ile Gly Leu Ser Ser His Leu His Thr Pro MetTyr Tyr 50 55 60 Phe Leu Ser Ser Leu Ser Phe Ile Asp Phe Cys Gln Ser ThrVal Val 65 70 75 80 Thr Pro Lys Met Leu Val Asn Phe Val Thr Glu Lys AsnIle Ile Ser 85 90 95 Tyr Pro Gly Cys Met Thr Gln Leu Tyr Phe Phe Leu IlePhe Ala Ile 100 105 110 Ala Glu Cys Tyr Ile Leu Ala Ala Met Ala Tyr AspArg Tyr Val Ala 115 120 125 Ile Cys Asn Pro Leu Leu Tyr Asn Val Thr MetSer Tyr Gln Ile Tyr 130 135 140 Ile Phe Leu Ile Ser Gly Val Tyr Ile IleGly Val Ile Cys Ala Ser 145 150 155 160 Ala His Thr Gly Phe Met Val ArgIle Arg Phe Cys Lys Leu Asp Val 165 170 175 Ile Asn His Tyr Phe Cys AspLeu Leu Pro Leu Leu Lys Leu Ala Cys 180 185 190 Ser Asn Thr Tyr Ile AsnGlu Met Leu Ile Leu Phe Phe Gly Thr Leu 195 200 205 Asn Ile Phe Val ProIle Leu Thr Ile Ile Thr Ser Tyr Ile Phe Ile 210 215 220 Ile Ala Ser IleLeu Arg Ile Arg Ser Thr Glu Gly Arg Ser Lys Ala 225 230 235 240 Phe SerThr Cys Ser Ser His Ile Leu Ala Val Ala Val Phe Phe Gly 245 250 255 SerLeu Ala Phe Met Tyr Leu Gln Pro Ser Ser Val Ser Ser Met Asp 260 265 270Gln Gly Lys Val Ser Ser Val Phe Tyr Thr Ile Val Val Pro Met Leu 275 280285 Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Asp Val Ala Val Ala Leu 290295 300 Lys Lys Ile Ile Glu Arg Lys Thr Phe Met 305 310 83 307 PRT Musmusculus 83 Met Glu Ser Gly Asn Leu Ser Met Ile Ile Glu Phe Ile Leu ThrGly 1 5 10 15 Phe Pro Thr Lys Pro Glu Leu Gln Leu Pro Leu Phe Leu LeuPhe Leu 20 25 30 Gly Ile Tyr Leu Val Thr Val Leu Gly Asn Leu Gly Met IleIle Leu 35 40 45 Ile Val Leu Ser Ser Gly Leu His Thr Pro Met Tyr Phe PheLeu Ser 50 55 60 Ser Leu Ser Phe Ile Asp Leu Cys His Ser Thr Val Ile ThrPro Lys 65 70 75 80 Met Leu Leu Asn Phe Leu Leu Glu Glu Asn Ile Ile SerTyr Pro Glu 85 90 95 Cys Met Thr Gln Leu Tyr Phe Phe Ser Leu Phe Ala IleAla Glu Cys 100 105 110 His Met Leu Ala Val Met Ala Tyr Asp Arg Tyr ValAla Ile Cys Asn 115 120 125 Pro Leu Leu Tyr Lys Val Val Met Ser His HisVal Cys Phe Trp Phe 130 135 140 Thr Val Gly Val Tyr Thr Leu Gly Ile LeuGly Ser Ser Val His Thr 145 150 155 160 Gly Leu Met Leu Lys Leu Phe PheCys Lys Thr Asn Lys Ile Asn His 165 170 175 Tyr Phe Cys Asp Leu Phe ProLeu Leu Glu Leu Ser Cys Ser Ser Ile 180 185 190 Tyr Ile Asn Glu Leu LeuVal Leu Phe Leu Ser Ala Leu Asn Ile Leu 195 200 205 Thr Pro Ala Leu ThrIle Leu Met Ser Tyr Ile Leu Ile Ile Val Ser 210 215 220 Ile Leu Arg IleArg Ser Thr Glu Gly Arg Ser Lys Ala Phe Ser Thr 225 230 235 240 Cys SerSer His Ile Ser Ala Val Ala Leu Phe Tyr Gly Ser Ala Ala 245 250 255 PheThr Tyr Leu Gln Pro Ser Ser Val Ser Ser Met Asn Gln Gly Lys 260 265 270Val Ser Ser Val Phe Tyr Thr Thr Val Val Pro Met Leu Asn Pro Leu 275 280285 Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Ser Ser Ile Lys Lys Ile 290295 300 Leu Asn Arg 305 84 299 PRT Mus musculus 84 Met Phe Ser Leu GluAsn Ala Leu Tyr Ile Gln Ala Gly Leu Gly Val 1 5 10 15 Leu Ala Asn MetCys Leu Leu Val Phe Tyr Ile Phe Met Val Leu Gly 20 25 30 His Arg Pro LysPro Met Asp Leu Ile Ser Cys Gln Gln Thr Phe Ile 35 40 45 His Ile Met LeuPhe Phe Thr Ala Gly Asp Ile Leu His Thr Asp Ile 50 55 60 Phe Glu Ser MetAsn Ile Glu Asn Asp Phe Lys Cys Lys Thr Thr Phe 65 70 75 80 Tyr Ile CysArg Val Met Arg Gly Leu Ser Ile Cys Thr Thr Cys Leu 85 90 95 Leu Ser ValPhe Gln Ala Val Thr Ile Ser Pro Asn Thr Ser Leu Leu 100 105 110 Ala LysPhe Lys His Lys Leu Lys Lys Tyr Thr Ile Asn Ala Phe Phe 115 120 125 TyrIle Trp Ser Phe Asn Leu Ser Phe Ser Ser Asn Leu Ile Phe Tyr 130 135 140Val Gly Ala Tyr Thr Asn Val Ser Glu Thr Asn Gln Met Lys Val Thr 145 150155 160 Lys Tyr Cys Ser Leu Phe Pro Met Asn Tyr Ile Ile Arg Gly Leu Ile165 170 175 Leu Thr Val Thr Thr Ser Arg Asp Val Phe Leu Val Gly Val MetLeu 180 185 190 Ile Thr Ser Thr Tyr Met Val Ile Ile Leu Phe Arg His GlnArg Gln 195 200 205 Cys Lys His Leu His Ser Ile Arg His Leu Arg Ala SerPro Glu Lys 210 215 220 Arg Ala Thr Gln Thr Ile Leu Leu Leu Val Ile PhePhe Val Val Met 225 230 235 240 Tyr Trp Val Asp Phe Ile Ile Ser Ser ThrSer Val Leu Leu Trp Met 245 250 255 Tyr Asp Pro Val Ile Leu Thr Val GlnLys Phe Val Met Asn Ala Tyr 260 265 270 Pro Thr Ile Thr Pro Leu Val GlnIle Ser Ser Asp Asn Arg Ile Ile 275 280 285 Asn Leu Leu Lys Asn Leu GlnSer Lys Cys His 290 295 85 1967 PRT Homo sapiens 85 Met Gln Leu Cys IlePhe Cys Cys Cys Cys Ile Leu Phe Tyr Phe Asp 1 5 10 15 Leu Tyr Asp PheGly Arg Gly Tyr Asp Phe Thr Ile Gln Glu Asn Gly 20 25 30 Leu Gln Ile AspGln Pro Pro Glu Ile Gly Asn Ile Ser Ile Val Arg 35 40 45 Ile Ile Ile MetLys Asn Asp Asn Ala Glu Gly Ile Ile Glu Phe Asp 50 55 60 Pro Lys Tyr ThrAla Phe Glu Val Glu Glu Asp Val Gly Leu Ile Met 65 70 75 80 Ile Pro ValVal Arg Leu His Gly Thr Tyr Gly Tyr Val Thr Ala Asp 85 90 95 Phe Ile SerGln Ser Ser Ser Ala Ser Pro Gly Gly Val Asp Tyr Ile 100 105 110 Leu HisGly Ser Thr Val Thr Phe Gln His Gly Gln Asn Leu Ser Phe 115 120 125 IleAsn Ile Ser Ile Ile Asp Asp Asn Glu Ser Glu Phe Glu Glu Pro 130 135 140Ile Glu Ile Leu Leu Thr Gly Ala Thr Gly Gly Ala Val Leu Gly Arg 145 150155 160 His Leu Val Ser Arg Ile Ile Ile Ala Lys Ser Asp Ser Pro Phe Gly165 170 175 Val Ile Arg Phe Leu Asn Gln Ser Lys Ile Ser Ile Ala Asn ProAsn 180 185 190 Ser Thr Met Ile Leu Ser Leu Val Leu Glu Arg Thr Gly GlyLeu Leu 195 200 205 Gly Glu Ile Gln Val Asn Trp Glu Thr Val Gly Pro AsnSer Gln Glu 210 215 220 Ala Leu Leu Pro Gln Asn Arg Asp Ile Ala Asp ProVal Ser Gly Leu 225 230 235 240 Phe Tyr Phe Gly Glu Gly Glu Gly Gly ValArg Thr Ile Ile Leu Thr 245 250 255 Ile Tyr Pro His Glu Glu Ile Glu ValGlu Glu Thr Phe Ile Ile Lys 260 265 270 Leu His Leu Val Lys Gly Glu AlaLys Leu Asp Ser Arg Ala Lys Asp 275 280 285 Val Thr Leu Thr Ile Gln GluPhe Gly Asp Pro Asn Gly Val Val Gln 290 295 300 Phe Ala Pro Glu Thr LeuSer Lys Lys Thr Tyr Ser Glu Pro Leu Ala 305 310 315 320 Leu Glu Gly ProLeu Leu Ile Thr Phe Phe Val Arg Arg Val Lys Gly 325 330 335 Thr Phe GlyGlu Ile Met Val Tyr Trp Glu Leu Ser Ser Glu Phe Asp 340 345 350 Ile ThrGlu Asp Phe Leu Ser Thr Ser Gly Phe Phe Thr Ile Ala Asp 355 360 365 GlyGlu Ser Glu Ala Ser Phe Asp Val His Leu Leu Pro Asp Glu Val 370 375 380Pro Glu Ile Glu Glu Asp Tyr Val Ile Gln Leu Val Ser Val Glu Gly 385 390395 400 Gly Ala Glu Leu Asp Leu Glu Lys Ser Ile Thr Trp Phe Ser Val Tyr405 410 415 Ala Asn Asp Asp Pro His Gly Val Phe Ala Leu Tyr Ser Asp ArgGln 420 425 430 Ser Ile Leu Ile Gly Gln Asn Leu Ile Arg Ser Ile Gln IleAsn Ile 435 440 445 Thr Arg Leu Ala Gly Thr Phe Gly Asp Val Ala Val GlyLeu Arg Ile 450 455 460 Ser Ser Asp His Lys Glu Gln Pro Ile Val Thr GluAsn Ala Glu Arg 465 470 475 480 Gln Leu Val Val Lys Asp Gly Ala Thr TyrLys Val Asp Val Val Pro 485 490 495 Ile Lys Asn Gln Val Phe Leu Ser LeuGly Ser Asn Phe Thr Leu Gln 500 505 510 Leu Val Thr Val Met Leu Val GlyGly Arg Phe Tyr Gly Met Pro Thr 515 520 525 Ile Leu Gln Glu Ala Lys SerAla Val Leu Pro Val Ser Glu Lys Ala 530 535 540 Ala Asn Ser Gln Val GlyPhe Glu Ser Thr Ala Phe Gln Leu Met Asn 545 550 555 560 Ile Thr Ala GlyThr Ser His Val Met Ile Ser Arg Arg Gly Thr Tyr 565 570 575 Gly Ala LeuSer Val Ala Trp Thr Thr Gly Tyr Ala Pro Gly Leu Glu 580 585 590 Ile ProGlu Phe Ile Val Val Gly Asn Met Thr Pro Thr Leu Gly Ser 595 600 605 LeuSer Phe Ser His Gly Glu Gln Arg Lys Gly Val Phe Leu Trp Thr 610 615 620Phe Pro Ser Pro Gly Trp Pro Glu Ala Phe Val Leu His Leu Ser Gly 625 630635 640 Val Gln Ser Ser Ala Pro Gly Gly Ala Gln Leu Arg Ser Gly Phe Ile645 650 655 Val Ala Glu Ile Glu Pro Met Gly Val Phe Gln Phe Ser Thr SerSer 660 665 670 Arg Asn Ile Ile Val Ser Glu Asp Thr Gln Met Ile Arg LeuHis Val 675 680 685 Gln Arg Leu Phe Gly Phe His Ser Asp Leu Ile Lys ValSer Tyr Gln 690 695 700 Thr Thr Ala Gly Ser Ala Lys Pro Leu Glu Asp PheGlu Pro Val Gln 705 710 715 720 Asn Gly Glu Leu Phe Phe Gln Lys Phe GlnThr Glu Val Asp Phe Glu 725 730 735 Ile Thr Ile Ile Asn Asp Gln Leu SerGlu Ile Glu Glu Phe Phe Tyr 740 745 750 Ile Asn Leu Thr Ser Val Glu IleArg Gly Leu Gln Lys Phe Asp Val 755 760 765 Asn Trp Ser Pro Arg Leu AsnLeu Asp Phe Ser Val Ala Val Ile Thr 770 775 780 Ile Leu Asp Asn Asp AspLeu Ala Gly Met Asp Ile Ser Phe Pro Glu 785 790 795 800 Thr Thr Val AlaVal Ala Val Asp Thr Thr Leu Ile Pro Val Glu Thr 805 810 815 Glu Ser ThrThr Tyr Leu Ser Thr Ser Lys Thr Thr Thr Ile Leu Gln 820 825 830 Pro ThrAsn Val Val Ala Ile Val Thr Glu Ala Thr Gly Val Ser Ala 835 840 845 IlePro Glu Lys Leu Val Thr Leu His Gly Thr Pro Ala Val Ser Glu 850 855 860Lys Pro Asp Val Ala Thr Val Thr Ala Asn Val Ser Ile His Gly Thr 865 870875 880 Phe Ser Leu Gly Pro Ser Ile Val Tyr Ile Glu Glu Glu Met Lys Asn885 890 895 Gly Thr Phe Asn Thr Ala Glu Val Leu Ile Arg Arg Thr Gly GlyPhe 900 905 910 Thr Gly Asn Val Ser Ile Thr Val Lys Thr Phe Gly Glu ArgCys Ala 915 920 925 Gln Met Glu Pro Asn Ala Leu Pro Phe Arg Gly Ile TyrGly Ile Ser 930 935 940 Asn Leu Thr Trp Ala Val Glu Glu Glu Asp Phe GluGlu Gln Thr Leu 945 950 955 960 Thr Leu Ile Phe Leu Asp Gly Glu Arg GluArg Lys Val Ser Val Gln 965 970 975 Ile Leu Asp Asp Asp Glu Pro Glu GlyGln Glu Phe Phe Tyr Val Phe 980 985 990 Leu Thr Asn Pro Gln Gly Gly AlaGln Ile Val Glu Gly Lys Asp Asp 995 1000 1005 Thr Gly Phe Ala Ala PheAla Met Val Ile Ile Thr Gly Ser Asp 1010 1015 1020 Leu His Asn Gly IleIle Gly Phe Ser Glu Glu Ser Gln Ser Gly 1025 1030 1035 Leu Glu Leu ArgGlu Gly Ala Val Met Arg Arg Leu His Leu Ile 1040 1045 1050 Val Thr ArgGln Pro Asn Arg Ala Phe Glu Asp Val Lys Val Phe 1055 1060 1065 Trp ArgVal Thr Leu Asn Lys Thr Val Val Val Leu Gln Lys Asp 1070 1075 1080 GlyVal Asn Leu Met Glu Glu Leu Gln Ser Val Ser Gly Thr Thr 1085 1090 1095Thr Cys Thr Met Gly Gln Thr Lys Cys Phe Ile Ser Ile Glu Leu 1100 11051110 Lys Pro Glu Lys Val Pro Gln Val Glu Val Tyr Phe Phe Val Glu 11151120 1125 Leu Tyr Glu Ala Thr Ala Gly Ala Ala Ile Asn Asn Ser Ala Arg1130 1135 1140 Phe Ala Gln Ile Lys Ile Leu Glu Ser Asp Glu Ser Gln SerLeu 1145 1150 1155 Val Tyr Phe Ser Val Gly Ser Arg Leu Ala Val Ala HisLys Lys 1160 1165 1170 Ala Thr Leu Ile Ser Leu Gln Val Ala Arg Asp SerGly Thr Gly 1175 1180 1185 Leu Met Met Ser Val Asn Phe Ser Thr Gln GluLeu Arg Ser Ala 1190 1195 1200 Glu Thr Ile Gly Arg Thr Ile Ile Ser ProAla Ile Ser Gly Lys 1205 1210 1215 Asp Phe Val Ile Thr Glu Gly Thr LeuVal Phe Glu Pro Gly Gln 1220 1225 1230 Arg Ser Thr Val Leu Asp Val IleLeu Thr Pro Glu Thr Gly Ser 1235 1240 1245 Leu Asn Ser Phe Pro Lys ArgPhe Gln Ile Val Leu Phe Asp Pro 1250 1255 1260 Lys Gly Gly Ala Arg IleAsp Lys Val Tyr Gly Thr Ala Asn Ile 1265 1270 1275 Thr Leu Val Ser AspAla Asp Ser Gln Ala Ile Trp Gly Leu Ala 1280 1285 1290 Asp Gln Leu HisGln Pro Val Asn Asp Asp Ile Leu Asn Arg Val 1295 1300 1305 Leu His ThrIle Ser Met Lys Val Ala Thr Glu Asn Thr Asp Glu 1310 1315 1320 Gln LeuSer Ala Met Met His Leu Ile Glu Lys Ile Thr Thr Glu 1325 1330 1335 GlyLys Ile Gln Ala Phe Ser Val Ala Ser Arg Thr Leu Phe Tyr 1340 1345 1350Glu Ile Leu Cys Ser Leu Ile Asn Pro Lys Arg Lys Asp Thr Arg 1355 13601365 Gly Phe Ser His Phe Ala Glu Val Thr Glu Asn Phe Ala Phe Ser 13701375 1380 Leu Leu Thr Asn Val Thr Cys Gly Ser Pro Gly Glu Lys Ser Lys1385 1390 1395 Thr Ile Leu Asp Ser Cys Pro Tyr Leu Ser Ile Leu Ala LeuHis 1400 1405 1410 Trp Tyr Pro Gln Gln Ile Asn Gly His Lys Phe Glu GlyLys Glu 1415 1420 1425 Gly Asp Tyr Ile Arg Ile Pro Glu Arg Leu Leu AspVal Gln Asp 1430 1435 1440 Ala Glu Ile Met Ala Gly Lys Ser Thr Cys LysLeu Val Gln Phe 1445 1450 1455 Thr Glu Tyr Ser Ser Gln Gln Trp Phe IleSer Gly Asn Asn Leu 1460 1465 1470 Pro Thr Leu Lys Asn Lys Val Leu SerLeu Ser Val Lys Gly Gln 1475 1480 1485 Ser Ser Gln Leu Leu Thr Asn AspAsn Glu Val Leu Tyr Arg Ile 1490 1495 1500 Tyr Ala Ala Glu Pro Arg IleIle Pro Gln Thr Ser Leu Cys Leu 1505 1510 1515 Leu Trp Asn Gln Ala AlaAla Ser Trp Leu Ser Asp Ser Gln Phe 1520 1525 1530 Cys Lys Val Ile GluGlu Thr Ala Asp Tyr Val Glu Cys Ala Cys 1535 1540 1545 Ser His Met SerVal Tyr Ala Val Tyr Ala Arg Thr Asp Asn Leu 1550 1555 1560 Ser Ser TyrAsn Glu Ala Phe Phe Thr Ser Gly Phe Ile Cys Ile 1565 1570 1575 Ser GlyLeu Cys Leu Ala Val Leu Ser His Ile Phe Cys Ala Arg 1580 1585 1590 TyrSer Met Phe Ala Ala Lys Leu Leu Thr His Met Met Ala Ala 1595 1600 1605Ser Leu Gly Thr Gln Ile Leu Phe Leu Ala Ser Ala Tyr Ala Ser 1610 16151620 Pro Gln Leu Ala Glu Glu Ser Cys Ser Ala Met Ala Ala Val Thr 16251630 1635 His Tyr Leu Tyr Leu Cys Gln Phe Ser Trp Met Leu Ile Gln Ser1640 1645 1650 Val Asn Phe Trp Tyr Val Leu Val Met Asn Asp Glu His ThrGlu 1655 1660 1665 Arg Arg Tyr Leu Leu Phe Phe Leu Leu Ser Trp Gly LeuPro Ala 1670 1675 1680 Phe Val Val Ile Leu Leu Ile Val Ile Leu Lys GlyIle Tyr His 1685 1690 1695 Gln Ser Met Ser Gln Ile Tyr Gly Leu Ile HisGly Asp Leu Cys 1700 1705 1710 Phe Ile Pro Asn Val Tyr Ala Ala Leu PheThr Ala Ala Leu Val 1715 1720 1725 Pro Leu Thr Cys Leu Val Val Val PheVal Val Phe Ile His Ala 1730 1735 1740 Tyr Gln Val Lys Pro Gln Trp LysAla Tyr Asp Asp Val Phe Arg 1745 1750 1755 Gly Arg Thr Asn Ala Ala GluIle Pro Leu Ile Leu Tyr Leu Phe 1760 1765 1770 Ala Leu Ile Ser Val ThrTrp Leu Trp Gly Gly Leu His Met Ala 1775 1780 1785 Tyr Arg His Phe TrpMet Leu Val Leu Phe Val Ile Phe Asn Ser 1790 1795 1800 Leu Gln Gly LeuTyr Val Phe Met Val Tyr Phe Ile Leu His Asn 1805 1810 1815 Gln Met CysCys Pro Met Lys Ala Ser Tyr Thr Val Glu Met Asn 1820 1825 1830 Gly HisPro Gly Pro Ser Thr Ala Phe Phe Thr Pro Gly Ser Gly 1835 1840 1845 MetPro Pro Ala Gly Gly Glu Ile Ser Lys Ser Thr Gln Asn Leu 1850 1855 1860Ile Gly Ala Met Glu Glu Val Pro Pro Asp Trp Glu Arg Ala Ser 1865 18701875 Phe Gln Gln Gly Ser Gln Ala Ser Pro Asp Leu Lys Pro Ser Pro 18801885 1890 Gln Asn Gly Ala Thr Phe Pro Ser Ser Gly Gly Tyr Gly Gln Gly1895 1900 1905 Ser Leu Ile Ala Asp Glu Glu Ser Gln Glu Phe Asp Asp LeuIle 1910 1915 1920 Phe Ala Leu Lys Thr Gly Ala Gly Leu Ser Val Ser AspAsn Glu 1925 1930 1935 Ser Gly Gln Gly Ser Gln Glu Gly Gly Thr Leu ThrAsp Ser Gln 1940 1945 1950 Ile Val Glu Leu Arg Arg Ile Pro Ile Ala AspThr His Leu 1955 1960 1965 86 259 PRT Homo sapiens 86 Gly Asn Ile LeuVal Ile Trp Val Ile Cys Arg His Lys Arg Met Arg 1 5 10 15 Thr Pro ThrAsn Tyr Phe Ile Cys Asn Leu Ala Val Ala Asp Leu Leu 20 25 30 Phe Cys LeuThr Cys Pro Pro Trp Met Leu Tyr Tyr Phe His Trp Gly 35 40 45 His His HisTrp Pro Phe Gly Arg Ala Met Cys Lys Ile Trp Thr Tyr 50 55 60 Phe Phe TyrMet Cys Cys Tyr Ala Ser Ile Phe Phe Leu Cys Cys Ile 65 70 75 80 Ser IleAsp Arg Tyr Trp Ala Ile Cys His Pro Met Arg Tyr Arg Arg 85 90 95 Arg MetThr Arg Pro Arg His Ala Trp Val Met Cys Leu Val Ile Trp 100 105 110 ValLeu Ala Phe Leu Trp Ser Leu Pro Pro Leu Met Phe Trp Trp Cys 115 120 125Tyr Thr His Glu Cys Pro Asn His Trp Asn Asn Cys Asn His Thr Trp 130 135140 Cys Phe Ile Asp Trp Pro His Glu Ser Trp His His Trp Trp Thr Trp 145150 155 160 Trp Arg Tyr Tyr Tyr Ile Cys Ser Cys Ile Val Gly Phe Tyr IlePro 165 170 175 Leu Leu Val Met Cys Phe Cys Tyr Cys Arg Ile Tyr Arg ThrLeu Trp 180 185 190 Lys Ala Ala Lys Met Leu Cys Val Val Val Val Val PhePhe Val Cys 195 200 205 Trp Leu Pro Tyr His Ile Phe Met Phe Met Asp ThrPhe Cys Met His 210 215 220 Trp Trp Met Ile Trp Thr Cys Glu Leu Glu CysVal Ile Pro Trp Ala 225 230 235 240 Tyr Gln Ile Cys Val Trp Leu Ala TyrVal Asn Cys Cys Leu Asn Pro 245 250 255 Ile Ile Tyr 87 26 PRT Homosapiens 87 Leu Ser Phe Thr Gly Leu Thr Cys Ile Val Ser Leu Val Ala LeuThr 1 5 10 15 Gly Asn Ala Val Val Leu Trp Leu Leu Gly 20 25 88 26 PRTHomo sapiens 88 Ile Tyr Ile Leu Asn Leu Val Ala Ala Asp Phe Leu Phe LeuCys Phe 1 5 10 15 Gln Ile Ile Asn Cys Leu Val Tyr Leu Ser 20 25 89 22PRT Homo sapiens 89 Phe Phe Thr Thr Val Met Thr Cys Ala Tyr Leu Ala GlyLeu Ser Met 1 5 10 15 Leu Ser Thr Val Ser Thr 20 90 19 PRT Homo sapiens90 Leu Ser Ala Val Val Cys Val Leu Leu Trp Ala Leu Ser Leu Leu Leu 1 510 15 Ser Ile Leu 91 23 PRT Homo sapiens 91 Phe Ile Thr Ala Ala Trp LeuIle Phe Leu Phe Met Val Leu Cys Gly 1 5 10 15 Ser Ser Leu Ala Leu LeuVal 20 92 21 PRT Homo sapiens 92 Leu Tyr Leu Thr Ile Leu Leu Thr Val LeuVal Phe Leu Leu Cys Gly 1 5 10 15 Leu Pro Phe Gly Ile 20 93 20 PRT Homosapiens 93 Val Ser Val Val Leu Ser Ser Leu Asn Ser Ser Ala Asn Pro IleIle 1 5 10 15 Tyr Phe Phe Val 20 94 13 PRT Homo sapiens 94 Leu Ser ThrVal Ser Thr Glu Arg Cys Leu Ser Val Leu 1 5 10 95 13 PRT Homo sapiens 95Tyr Phe Phe Val Gly Ser Phe Arg Lys Gln Trp Arg Leu 1 5 10 96 14 PRTHomo sapiens 96 Leu Ser Leu Leu Leu Ser Ile Leu Glu Gly Lys Phe Cys Gly1 5 10 97 14 PRT Homo sapiens 97 Cys Gly Phe Leu Phe Ser Asp Gly Asp SerGly Trp Cys Gln 1 5 10 98 10 PRT Homo sapiens 98 Leu Glu Leu Ser Gly SerArg Leu Glu Gln 1 5 10 99 14 PRT Homo sapiens 99 Val Leu Ser Ser Leu AsnSer Ser Ala Asn Pro Ile Ile Tyr 1 5 10 100 16 PRT Homo sapiens 100 PheMet Val Leu Cys Gly Ser Ser Leu Ala Leu Leu Val Arg Ile Leu 1 5 10 15101 16 PRT Homo sapiens 101 Leu Cys Gly Ser Arg Gly Leu Pro Leu Thr ArgLeu Tyr Leu Thr Ile 1 5 10 15 102 16 PRT Homo sapiens 102 Val Phe LeuLeu Cys Gly Leu Pro Phe Gly Ile Gln Trp Phe Leu Ile 1 5 10 15 103 32 PRTHomo sapiens 103 Cys Gly Ser Arg Gly Leu Pro Leu Thr Arg Leu Tyr Leu ThrIle Leu 1 5 10 15 Leu Thr Val Leu Val Phe Leu Leu Cys Gly Leu Pro PheGly Ile Gln 20 25 30 104 27 PRT Homo sapiens 104 Thr Cys Ala Tyr Leu AlaGly Leu Ser Met Leu Ser Thr Val Ser Thr 1 5 10 15 Glu Arg Cys Leu SerVal Leu Trp Pro Ile Trp 20 25 105 26 PRT Homo sapiens 105 Leu Pro LeuPhe Leu Leu Phe Leu Gly Ile Tyr Val Val Thr Val Val 1 5 10 15 Gly AsnLeu Gly Met Thr Thr Leu Ile Trp 20 25 106 23 PRT Homo sapiens 106 TyrPhe Leu Ser Ser Leu Ser Phe Ile Asp Phe Cys His Ser Thr Val 1 5 10 15Ile Thr Pro Lys Met Leu Val 20 107 24 PRT Homo sapiens 107 Cys Met ThrGln Leu Tyr Phe Phe Leu Val Phe Ala Ile Ala Glu Cys 1 5 10 15 His MetLeu Ala Ala Met Ala Tyr 20 108 19 PRT Homo sapiens 108 Ala Cys Phe SerLeu Ile Leu Gly Val Tyr Ile Ile Gly Leu Val Cys 1 5 10 15 Ala Ser Val109 30 PRT Homo sapiens 109 Leu Leu Ile Leu Cys Val Gly Ala Phe Asn IleLeu Val Pro Ser Leu 1 5 10 15 Thr Ile Leu Cys Ser Tyr Ile Phe Ile IleAla Ser Ile Leu 20 25 30 110 22 PRT Homo sapiens 110 His Met Leu Ala ValVal Ile Phe Phe Gly Ser Ala Ala Phe Met Tyr 1 5 10 15 Leu Gln Pro SerSer Ile 20 111 20 PRT Homo sapiens 111 Val Ser Ser Val Phe Tyr Thr IleIle Val Pro Met Leu Asn Pro Leu 1 5 10 15 Ile Tyr Ser Leu 20 112 13 PRTHomo sapiens 112 His Ser Thr Val Ile Thr Pro Lys Met Leu Val Asn Phe 1 510 113 13 PRT Homo sapiens 113 Leu Val Asn Phe Val Thr Glu Lys Asn IleIle Ser Tyr 1 5 10 114 13 PRT Homo sapiens 114 Tyr Ser Val Ile Ile SerAsn Lys Ala Cys Phe Ser Leu 1 5 10 115 13 PRT Homo sapiens 115 Asn ProLeu Ile Tyr Ser Leu Arg Asn Lys Asp Val His 1 5 10 116 13 PRT Homosapiens 116 Lys Asp Val His Val Ser Leu Lys Lys Met Leu Gln Arg 1 5 10117 14 PRT Homo sapiens 117 Gly Glu Asn Asn Ser Ser Val Thr Glu Phe IleLeu Ala Gly 1 5 10 118 14 PRT Homo sapiens 118 Phe Leu Ser Ser Leu SerPhe Ile Asp Phe Cys His Ser Thr 1 5 10 119 14 PRT Homo sapiens 119 GluLys Asn Ile Ile Ser Tyr Pro Glu Cys Met Thr Gln Leu 1 5 10 120 14 PRTHomo sapiens 120 Gln Pro Ser Ser Ile Ser Ser Met Asp Gln Gly Lys Val Ser1 5 10 121 13 PRT Homo sapiens 121 Met Ser Gly Glu Asn Asn Ser Ser ValThr Glu Phe Ile 1 5 10 122 14 PRT Homo sapiens 122 Met Ser Gly Glu AsnAsn Ser Ser Val Thr Glu Phe Ile Leu 1 5 10 123 16 PRT Homo sapiens 123Gly Val Tyr Ile Ile Gly Leu Val Cys Ala Ser Val His Thr Gly Cys 1 5 1015 124 27 PRT Homo sapiens 124 Leu Val Phe Ala Ile Ala Glu Cys His MetLeu Ala Ala Met Ala Tyr 1 5 10 15 Asp Arg Tyr Met Ala Ile Cys Ser ProLeu Leu 20 25 125 19 PRT Homo sapiens 125 Leu Leu Ile Ile Ile Phe PhePhe Tyr Leu Leu Glu Leu Val His Ala 1 5 10 15 Ala Ser Leu 126 20 PRTHomo sapiens 126 Phe Val Ser Gly Thr Glu Pro Glu Asp Gly Tyr Ser Thr ValThr Leu 1 5 10 15 Asn Val Ile Arg 20 127 21 PRT Homo sapiens 127 Ile AspSer Asp Pro Asp Gly Asp Leu Ala Phe Thr Ser Gly Asn Ile 1 5 10 15 ThrPhe Glu Ile Gly 20 128 19 PRT Homo sapiens 128 Ala Phe Ser Val Ser ValLeu Ser Val Ser Ser Gly Ser Leu Gly Ala 1 5 10 15 His Ile Asn 129 18 PRTHomo sapiens 129 Lys Val Glu Glu Ala Thr Gln Asn Ile Thr Leu Ser Ile IleArg Leu 1 5 10 15 Lys Gly 130 25 PRT Homo sapiens 130 Ala Thr Gln GlyArg Asp Tyr Ile Pro Ala Ser Gly Phe Ala Leu Phe 1 5 10 15 Gly Ala AsnGln Ser Glu Ala Thr Ile 20 25 131 19 PRT Homo sapiens 131 Glu Ser ValPhe Ile Glu Leu Leu Asn Ser Thr Leu Val Ala Lys Val 1 5 10 15 Gln SerArg 132 13 PRT Homo sapiens 132 Gly Ile Phe Ile Phe Ser Glu Lys Asn ArgPro Val Lys 1 5 10 133 13 PRT Homo sapiens 133 Lys Val Gln Ser Arg SerSer Lys Tyr Pro Leu Val Cys 1 5 10 134 14 PRT Homo sapiens 134 Glu SerLeu Phe Val Ser Gly Thr Glu Pro Glu Asp Gly Tyr 1 5 10 135 14 PRT Homosapiens 135 Leu Phe Val Ser Gly Thr Glu Pro Glu Asp Gly Tyr Ser Thr 1 510 136 14 PRT Homo sapiens 136 His Trp Asn Ile Asp Ser Asp Pro Asp GlyAsp Leu Ala Phe 1 5 10 137 14 PRT Homo sapiens 137 Leu Val Ser Tyr AlaThr Leu Asp Asp Met Glu Lys Pro Pro 1 5 10 138 14 PRT Homo sapiens 138Ala Thr Ile Ala Ile Ser Ile Leu Asp Asp Asp Glu Pro Glu 1 5 10 139 14PRT Homo sapiens 139 Ala Phe Thr Ser Gly Asn Ile Thr Phe Glu Ile Gly GlnThr 1 5 10 140 14 PRT Homo sapiens 140 Gly Gln Thr Ser Ala Asn Ile ThrVal Glu Ile Leu Pro Asp 1 5 10 141 14 PRT Homo sapiens 141 Leu Gly AlaHis Ile Asn Ala Thr Leu Thr Val Leu Ala Ser 1 5 10 142 14 PRT Homosapiens 142 Glu Glu Ala Thr Gln Asn Ile Thr Leu Ser Ile Ile Arg Leu 1 510 143 14 PRT Homo sapiens 143 Ala Leu Phe Gly Ala Asn Gln Ser Glu AlaThr Ile Ala Ile 1 5 10 144 14 PRT Homo sapiens 144 Phe Ile Glu Leu LeuAsn Ser Thr Leu Val Ala Lys Val Gln 1 5 10 145 16 PRT Homo sapiens 145Ile Thr Phe Glu Ile Gly Gln Thr Ser Ala Asn Ile Thr Val Glu Ile 1 5 1015 146 16 PRT Homo sapiens 146 Leu Ser Val Ser Ser Gly Ser Leu Gly AlaHis Ile Asn Ala Thr Leu 1 5 10 15 147 16 PRT Homo sapiens 147 Ser SerGly Ser Leu Gly Ala His Ile Asn Ala Thr Leu Thr Val Leu 1 5 10 15 148 16PRT Homo sapiens 148 Gly Phe Ala Leu Phe Gly Ala Asn Gln Ser Glu Ala ThrIle Ala Ile 1 5 10 15 149 1615 PRT Homo sapiens 149 Asp Ala Trp Ala AspAla Trp Ala Leu Tyr Thr Cys Ala Thr Leu Cys 1 5 10 15 Leu Lys Glu GlnAla Cys Ser Ala Phe Ser Phe Phe Ser Ala Ser Glu 20 25 30 Gly Pro Gln CysPhe Trp Met Thr Ser Trp Ile Ser Pro Ala Val Asn 35 40 45 Asn Ser Asp PheTrp Thr Tyr Arg Lys Asn Met Thr Arg Val Ala Ser 50 55 60 Leu Phe Ser GlyGln Ala Val Ala Gly Ser Asp Tyr Glu Pro Val Thr 65 70 75 80 Arg Gln TrpAla Ile Met Gln Glu Gly Asp Glu Phe Ala Asn Leu Thr 85 90 95 Val Ser IleLeu Pro Asp Asp Phe Pro Glu Met Asp Glu Ser Phe Leu 100 105 110 Ile SerLeu Leu Glu Val His Leu Met Asn Ile Ser Ala Ser Leu Lys 115 120 125 AsnGln Pro Thr Ile Gly Gln Pro Asn Ile Ser Thr Val Val Ile Ala 130 135 140Leu Asn Gly Asp Ala Phe Gly Val Phe Val Ile Tyr Ser Ile Ser Pro 145 150155 160 Asn Thr Ser Glu Asp Gly Leu Phe Val Glu Val Gln Glu Gln Pro Gln165 170 175 Thr Leu Val Glu Leu Met Ile His Arg Thr Gly Gly Ser Leu GlyGln 180 185 190 Val Ala Val Glu Trp Arg Val Val Gly Gly Thr Ala Thr GluGly Leu 195 200 205 Asp Phe Ile Gly Ala Gly Glu Ile Leu Thr Phe Ala GluGly Glu Thr 210 215 220 Lys Lys Thr Val Ile Leu Thr Ile Leu Asp Asp SerGlu Pro Glu Asp 225 230 235 240 Asp Glu Ser Ile Ile Val Ser Leu Val TyrThr Glu Gly Gly Ser Arg 245 250 255 Ile Leu Pro Ser Ser Asp Thr Val ArgVal Asn Ile Leu Ala Asn Asp 260 265 270 Asn Val Ala Gly Ile Val Ser PheGln Thr Ala Ser Arg Ser Val Ile 275 280 285 Gly His Glu Gly Glu Ile LeuGln Phe His Val Ile Arg Thr Phe Pro 290 295 300 Gly Arg Gly Asn Val ThrVal Asn Trp Lys Ile Ile Gly Gln Asn Leu 305 310 315 320 Glu Leu Asn PheAla Asn Phe Ser Gly Gln Leu Phe Phe Pro Glu Gly 325 330 335 Ser Leu AsnThr Thr Leu Phe Val His Leu Leu Asp Asp Asn Ile Pro 340 345 350 Glu GluLys Glu Val Tyr Gln Val Ile Leu Tyr Asp Val Arg Thr Gln 355 360 365 GlyVal Pro Pro Ala Gly Ile Ala Leu Leu Asp Ala Gln Gly Tyr Ala 370 375 380Ala Val Leu Thr Val Glu Ala Ser Asp Glu Pro His Gly Val Leu Asn 385 390395 400 Phe Ala Leu Ser Ser Arg Phe Val Leu Leu Gln Glu Ala Asn Ile Thr405 410 415 Ile Gln Leu Phe Ile Asn Arg Glu Phe Gly Ser Leu Gly Ala IleAsn 420 425 430 Val Thr Tyr Thr Thr Val Pro Gly Met Leu Ser Leu Lys AsnGln Thr 435 440 445 Val Gly Asn Leu Ala Glu Pro Glu Val Asp Phe Val ProIle Ile Gly 450 455 460 Phe Leu Ile Leu Glu Glu Gly Glu Thr Ala Ala AlaIle Asn Ile Thr 465 470 475 480 Ile Leu Glu Asp Asp Val Pro Glu Leu GluGlu Tyr Phe Leu Val Asn 485 490 495 Leu Thr Tyr Val Gly Leu Thr Met AlaAla Ser Thr Ser Phe Pro Pro 500 505 510 Arg Leu Asp Ser Glu Gly Leu ThrAla Gln Val Ile Ile Asp Ala Asn 515 520 525 Asp Gly Ala Arg Gly Val IleGlu Trp Gln Gln Ser Arg Phe Glu Val 530 535 540 Asn Glu Thr His Gly SerLeu Thr Leu Val Ala Gln Arg Ser Arg Glu 545 550 555 560 Pro Leu Gly HisVal Ser Leu Phe Val Tyr Ala Gln Asn Leu Glu Ala 565 570 575 Gln Val GlyLeu Asp Tyr Ile Phe Thr Pro Met Ile Leu His Phe Ala 580 585 590 Asp GlyGlu Arg Tyr Lys Asn Val Asn Ile Met Ile Leu Asp Asp Asp 595 600 605 IlePro Glu Gly Asp Glu Lys Phe Gln Leu Ile Leu Thr Asn Pro Ser 610 615 620Pro Gly Leu Glu Leu Gly Lys Asn Thr Ile Ala Leu Ile Ile Val Leu 625 630635 640 Ala Asn Asp Asp Gly Pro Gly Val Leu Ser Phe Asn Asn Ser Glu His645 650 655 Phe Phe Leu Arg Glu Pro Thr Ala Leu Tyr Val Gln Glu Ser ValAla 660 665 670 Val Leu Tyr Ile Val Arg Glu Pro Ala Gln Gly Leu Phe GlyThr Val 675 680 685 Thr Val Gln Phe Ile Val Thr Glu Val Asn Ser Ser AsnGlu Ser Lys 690 695 700 Asp Leu Thr Pro Ser Lys Gly Tyr Ile Val Leu GluGlu Gly Val Arg 705 710 715 720 Phe Lys Ala Leu Gln Ile Ser Ala Ile LeuAsp Thr Glu Pro Glu Met 725 730 735 Asp Glu Tyr Phe Val Cys Thr Leu PheAsn Pro Thr Gly Gly Ala Arg 740 745 750 Leu Gly Val His Val Gln Thr LeuIle Thr Val Leu Gln Asn Gln Ala 755 760 765 Pro Leu Gly Leu Phe Ser IleSer Ala Val Glu Asn Arg Ala Thr Ser 770 775 780 Ile Asp Ile Glu Glu AlaAsn Arg Thr Val Tyr Leu Asn Val Ser Arg 785 790 795 800 Thr Asn Gly IleAsp Leu Ala Val Ser Val Gln Trp Glu Thr Val Ser 805 810 815 Glu Thr AlaPhe Gly Met Arg Gly Met Asp Val Val Phe Ser Val Phe 820 825 830 Gln SerPhe Leu Asp Glu Ser Ala Ser Gly Trp Cys Phe Phe Thr Leu 835 840 845 GluAsn Leu Ile Tyr Gly Ile Met Leu Arg Lys Ser Ser Val Thr Val 850 855 860Tyr Arg Trp Gln Gly Ile Phe Ile Pro Val Glu Asp Leu Asn Ile Glu 865 870875 880 Asn Pro Lys Thr Cys Glu Ala Phe Asn Ile Gly Phe Ser Pro Tyr Phe885 890 895 Val Ile Thr His Glu Glu Arg Asn Glu Glu Lys Pro Ser Leu AsnSer 900 905 910 Val Phe Thr Phe Thr Ser Gly Phe Lys Leu Phe Leu Val GlnThr Ile 915 920 925 Ile Ile Leu Glu Ser Ser Gln Val Arg Tyr Phe Thr SerAsp Ser Gln 930 935 940 Asp Tyr Leu Ile Ile Ala Ser Gln Arg Asp Asp SerGlu Leu Thr Gln 945 950 955 960 Val Phe Arg Trp Asn Gly Gly Ser Phe ValLeu His Gln Lys Leu Pro 965 970 975 Val Arg Gly Val Leu Thr Val Ala LeuPhe Asn Lys Gly Gly Ser Val 980 985 990 Phe Leu Ala Ile Ser Gln Ala AsnAla Arg Leu Asn Ser Leu Leu Phe 995 1000 1005 Arg Trp Ser Gly Ser GlyPhe Ile Asn Phe Gln Glu Val Pro Val 1010 1015 1020 Ser Gly Thr Thr GluVal Glu Ala Leu Ser Ser Ala Asn Asp Ile 1025 1030 1035 Tyr Leu Ile PheAla Lys Asn Val Phe Leu Gly Asp Gln Asn Ser 1040 1045 1050 Ile Asp IlePhe Ile Trp Glu Met Gly Gln Ser Ser Phe Arg Tyr 1055 1060 1065 Phe GlnSer Val Asp Phe Ala Ala Val Asn Arg Ile His Ser Phe 1070 1075 1080 ThrPro Ala Ser Gly Ile Ala His Ile Leu Leu Ile Gly Gln Asp 1085 1090 1095Met Ser Ala Leu Tyr Cys Trp Asn Ser Glu Arg Asn Gln Phe Ser 1100 11051110 Phe Val Leu Glu Val Pro Ser Ala Tyr Asp Val Ala Ser Val Thr 11151120 1125 Val Lys Ser Leu Asn Ser Ser Lys Asn Leu Ile Ala Leu Val Gly1130 1135 1140 Ala His Ser His Ile Tyr Glu Leu Ala Tyr Ile Ser Ser HisSer 1145 1150 1155 Asp Phe Ile Pro Ser Ser Gly Glu Leu Ile Phe Glu ProGly Glu 1160 1165 1170 Arg Glu Ala Thr Ile Ala Val Asn Ile Leu Asp AspThr Val Pro 1175 1180 1185 Glu Lys Glu Glu Ser Phe Lys Val Gln Leu LysAsn Pro Lys Gly 1190 1195 1200 Gly Ala Glu Ile Gly Ile Asn Asp Ser ValThr Ile Thr Ile Leu 1205 1210 1215 Ser Asn Asp Asp Ala Tyr Gly Ile ValAla Phe Ala Gln Asn Ser 1220 1225 1230 Leu Tyr Lys Gln Val Glu Glu MetGlu Gln Asp Ser Leu Val Thr 1235 1240 1245 Leu Asn Val Glu Arg Leu LysGly Thr Tyr Gly Arg Ile Thr Ile 1250 1255 1260 Ala Trp Glu Ala Asp GlySer Ile Ser Asp Ile Phe Pro Thr Ser 1265 1270 1275 Gly Val Ile Leu PheThr Glu Gly Gln Val Leu Ser Thr Ile Thr 1280 1285 1290 Leu Thr Ile LeuAla Asp Asn Ile Pro Glu Leu Ser Glu Val Val 1295 1300 1305 Ile Val ThrLeu Thr Arg Ile Thr Thr Glu Gly Val Glu Asp Ser 1310 1315 1320 Tyr LysGly Ala Thr Ile Asp Gln Asp Arg Ser Lys Ser Val Ile 1325 1330 1335 ThrThr Leu Pro Asn Asp Ser Pro Phe Gly Leu Val Gly Trp Arg 1340 1345 1350Ala Ala Ser Val Phe Ile Arg Val Ala Glu Pro Lys Glu Asn Thr 1355 13601365 Thr Thr Leu Gln Leu Gln Ile Ala Arg Asp Lys Gly Leu Leu Gly 13701375 1380 Asp Ile Ala Ile His Leu Arg Ala Gln Pro Asn Phe Leu Leu His1385 1390 1395 Val Asp Asn Gln Ala Thr Glu Asn Glu Asp Tyr Val Leu GlnGlu 1400 1405 1410 Thr Ile Ile Ile Met Lys Glu Asn Ile Lys Glu Ala HisAla Glu 1415 1420 1425 Val Ser Ile Leu Pro Asp Asp Leu Pro Glu Leu GluGlu Gly Phe 1430 1435 1440 Ile Val Thr Ile Thr Glu Val Asn Leu Val AsnSer Asp Phe Ser 1445 1450 1455 Thr Gly Gln Pro Ser Val Arg Arg Pro GlyMet Glu Ile Ala Glu 1460 1465 1470 Ile Met Ile Glu Glu Asn Asp Asp ProArg Gly Ile Phe Met Phe 1475 1480 1485 His Val Thr Arg Gly Ala Gly GluVal Ile Thr Ala Tyr Glu Val 1490 1495 1500 Pro Pro Pro Leu Asn Val LeuGln Val Pro Val Val Arg Leu Ala 1505 1510 1515 Gly Ser Phe Gly Ala ValAsn Val Tyr Trp Lys Ala Ser Pro Asp 1520 1525 1530 Ser Ala Gly Leu GluAsp Phe Lys Pro Ser His Gly Ile Leu Glu 1535 1540 1545 Phe Ala Asp LysGln Val Thr Ala Met Ile Glu Ile Thr Ile Ile 1550 1555 1560 Asp Asp AlaGlu Phe Glu Leu Thr Glu Thr Phe Asn Ile Ser Leu 1565 1570 1575 Ile SerVal Ala Gly Gly Gly Arg Leu Gly Asp Asp Val Val Val 1580 1585 1590 ThrVal Val Ile Pro Gln Asn Asp Ser Pro Phe Gly Val Phe Gly 1595 1600 1605Phe Glu Glu Lys Thr Val Ser 1610 1615 150 1577 PRT Homo sapiens 150 MetThr Ser Trp Ile Ser Pro Ala Val Asn Asn Ser Asp Phe Trp Thr 1 5 10 15Tyr Arg Lys Asn Met Thr Arg Val Ala Ser Leu Phe Ser Gly Gln Ala 20 25 30Val Ala Gly Ser Asp Tyr Glu Pro Val Thr Arg Gln Trp Ala Ile Met 35 40 45Gln Glu Gly Asp Glu Phe Ala Asn Leu Thr Val Ser Ile Leu Pro Asp 50 55 60Asp Phe Pro Glu Met Asp Glu Ser Phe Leu Ile Ser Leu Leu Glu Val 65 70 7580 His Leu Met Asn Ile Ser Ala Ser Leu Lys Asn Gln Pro Thr Ile Gly 85 9095 Gln Pro Asn Ile Ser Thr Val Val Ile Ala Leu Asn Gly Asp Ala Phe 100105 110 Gly Val Phe Val Ile Tyr Asn Ile Ser Pro Asn Thr Ser Glu Asp Gly115 120 125 Leu Phe Val Glu Val Gln Glu Gln Pro Gln Thr Leu Val Glu LeuMet 130 135 140 Ile His Arg Thr Gly Gly Ser Leu Gly Gln Val Ala Val GluTrp Arg 145 150 155 160 Val Val Gly Gly Thr Ala Thr Glu Gly Leu Asp PheIle Gly Ala Gly 165 170 175 Glu Ile Leu Thr Phe Ala Glu Gly Glu Thr LysLys Thr Val Ile Leu 180 185 190 Thr Ile Leu Asp Asp Ser Glu Pro Glu AspAsp Glu Ser Ile Ile Val 195 200 205 Ser Leu Val Tyr Thr Glu Gly Gly SerArg Ile Leu Pro Ser Ser Asp 210 215 220 Thr Val Arg Val Asn Ile Leu AlaAsn Asp Asn Val Ala Gly Ile Val 225 230 235 240 Ser Phe Gln Thr Ala SerArg Ser Val Ile Gly His Glu Gly Glu Ile 245 250 255 Leu Gln Phe His ValIle Arg Thr Phe Pro Gly Arg Gly Asn Val Thr 260 265 270 Val Asn Trp LysIle Ile Gly Gln Asn Leu Glu Leu Asn Phe Ala Asn 275 280 285 Phe Ser GlyGln Leu Phe Phe Pro Glu Gly Ser Leu Asn Thr Thr Leu 290 295 300 Phe ValHis Leu Leu Asp Asp Asn Ile Pro Glu Glu Lys Glu Val Tyr 305 310 315 320Gln Val Ile Leu Tyr Asp Val Arg Thr Gln Gly Val Pro Pro Ala Gly 325 330335 Ile Ala Leu Leu Asp Ala Gln Gly Tyr Ala Ala Val Leu Thr Val Glu 340345 350 Ala Ser Asp Glu Pro His Gly Val Leu Asn Phe Ala Leu Ser Ser Arg355 360 365 Phe Val Leu Leu Gln Glu Ala Asn Ile Thr Ile Gln Leu Phe IleAsn 370 375 380 Arg Glu Phe Gly Ser Leu Gly Ala Ile Asn Val Thr Tyr ThrThr Val 385 390 395 400 Pro Gly Met Leu Ser Leu Lys Asn Gln Thr Val GlyAsn Leu Ala Glu 405 410 415 Pro Glu Val Asp Phe Val Pro Ile Ile Gly PheLeu Ile Leu Glu Glu 420 425 430 Gly Glu Thr Ala Ala Ala Ile Asn Ile ThrIle Leu Glu Asp Asp Val 435 440 445 Pro Glu Leu Glu Glu Tyr Phe Leu ValAsn Leu Thr Tyr Val Gly Leu 450 455 460 Thr Met Ala Ala Ser Thr Ser PhePro Pro Arg Leu Asp Ser Glu Gly 465 470 475 480 Leu Thr Ala Gln Val IleIle Asp Ala Asn Asp Gly Ala Arg Gly Val 485 490 495 Ile Glu Trp Gln GlnSer Arg Phe Glu Val Asn Glu Thr His Gly Ser 500 505 510 Leu Thr Leu ValAla Gln Arg Ser Arg Glu Pro Leu Gly His Val Ser 515 520 525 Leu Phe ValTyr Ala Gln Asn Leu Glu Ala Gln Val Gly Leu Asp Tyr 530 535 540 Ile PheThr Pro Met Ile Leu His Phe Ala Asp Gly Glu Arg Tyr Lys 545 550 555 560Asn Val Asn Ile Met Ile Leu Asp Asp Asp Ile Pro Glu Gly Asp Glu 565 570575 Lys Phe Gln Leu Ile Leu Thr Asn Pro Ser Pro Gly Leu Glu Leu Gly 580585 590 Lys Asn Thr Ile Ala Leu Ile Ile Val Leu Ala Asn Asp Asp Gly Pro595 600 605 Gly Val Leu Ser Phe Asn Asn Ser Glu His Phe Phe Leu Arg GluPro 610 615 620 Thr Ala Leu Tyr Val Gln Glu Ser Val Ala Val Leu Tyr IleVal Arg 625 630 635 640 Glu Pro Ala Gln Gly Leu Phe Gly Thr Val Thr ValGln Phe Ile Val 645 650 655 Thr Glu Val Asn Ser Ser Asn Glu Ser Lys AspLeu Thr Pro Ser Lys 660 665 670 Gly Tyr Ile Val Leu Glu Glu Gly Val ArgPhe Lys Ala Leu Gln Ile 675 680 685 Ser Ala Ile Leu Asp Thr Glu Pro GluMet Asp Glu Tyr Phe Val Cys 690 695 700 Thr Leu Phe Asn Pro Thr Gly GlyAla Arg Leu Gly Val His Val Gln 705 710 715 720 Thr Leu Ile Thr Val LeuGln Asn Gln Ala Pro Leu Gly Leu Phe Ser 725 730 735 Ile Ser Ala Val GluAsn Arg Ala Thr Ser Ile Asp Ile Glu Glu Ala 740 745 750 Asn Arg Thr ValTyr Leu Asn Val Ser Arg Thr Asn Gly Ile Asp Leu 755 760 765 Ala Val SerVal Gln Trp Glu Thr Val Ser Glu Thr Ala Phe Gly Met 770 775 780 Arg GlyMet Asp Val Val Phe Ser Val Phe Gln Ser Phe Leu Asp Glu 785 790 795 800Ser Ala Ser Gly Trp Cys Phe Phe Thr Leu Glu Asn Leu Ile Tyr Gly 805 810815 Ile Met Leu Arg Lys Ser Ser Val Thr Val Tyr Arg Trp Gln Gly Ile 820825 830 Phe Ile Pro Val Glu Asp Leu Asn Ile Glu Asn Pro Lys Thr Cys Glu835 840 845 Ala Phe Asn Ile Gly Phe Ser Pro Tyr Phe Val Ile Thr His GluGlu 850 855 860 Arg Asn Glu Glu Lys Pro Ser Leu Asn Ser Val Phe Thr PheThr Ser 865 870 875 880 Gly Phe Lys Leu Phe Leu Val Gln Thr Ile Ile IleLeu Glu Ser Ser 885 890 895 Gln Val Arg Tyr Phe Thr Ser Asp Ser Gln AspTyr Leu Ile Ile Ala 900 905 910 Ser Gln Arg Asp Asp Ser Glu Leu Thr GlnVal Phe Arg Trp Asn Gly 915 920 925 Gly Ser Phe Val Leu His Gln Lys LeuPro Val Arg Gly Val Leu Thr 930 935 940 Val Ala Leu Phe Asn Lys Gly GlySer Val Phe Leu Ala Ile Ser Gln 945 950 955 960 Ala Asn Ala Arg Leu AsnSer Leu Leu Phe Arg Trp Ser Gly Ser Gly 965 970 975 Phe Ile Asn Phe GlnGlu Val Pro Val Ser Gly Thr Thr Glu Val Glu 980 985 990 Ala Leu Ser SerAla Asn Asp Ile Tyr Leu Ile Phe Ala Glu Asn Val 995 1000 1005 Phe LeuGly Asp Gln Asn Ser Ile Asp Ile Phe Ile Trp Glu Met 1010 1015 1020 GlyGln Ser Ser Phe Arg Tyr Phe Gln Ser Val Asp Phe Ala Ala 1025 1030 1035Val Asn Arg Ile His Ser Phe Thr Pro Ala Ser Gly Ile Ala His 1040 10451050 Ile Leu Leu Ile Gly Gln Asp Met Ser Ala Leu Tyr Cys Trp Asn 10551060 1065 Ser Glu Arg Asn Gln Phe Ser Phe Val Leu Glu Val Pro Ser Ala1070 1075 1080 Tyr Asp Val Ala Ser Val Thr Val Lys Ser Leu Asn Ser SerLys 1085 1090 1095 Asn Leu Ile Ala Leu Val Gly Ala His Ser His Ile TyrGlu Leu 1100 1105 1110 Ala Tyr Ile Ser Ser His Ser Asp Phe Ile Pro SerSer Gly Glu 1115 1120 1125 Leu Ile Phe Glu Pro Gly Glu Arg Glu Ala ThrIle Ala Val Asn 1130 1135 1140 Ile Leu Asp Asp Thr Val Pro Glu Lys GluGlu Ser Phe Lys Val 1145 1150 1155 Gln Leu Lys Asn Pro Lys Gly Gly AlaGlu Ile Gly Ile Asn Asp 1160 1165 1170 Ser Val Thr Ile Thr Ile Leu SerAsn Asp Asp Ala Tyr Gly Ile 1175 1180 1185 Val Ala Phe Ala Gln Asn SerLeu Tyr Lys Gln Val Glu Glu Met 1190 1195 1200 Glu Gln Asp Ser Leu ValThr Leu Asn Val Glu Arg Leu Lys Gly 1205 1210 1215 Thr Tyr Gly Arg IleThr Ile Ala Trp Glu Ala Asp Gly Ser Ile 1220 1225 1230 Ser Asp Ile PhePro Thr Ser Gly Val Ile Leu Phe Thr Glu Gly 1235 1240 1245 Gln Val LeuSer Thr Ile Thr Leu Thr Ile Leu Ala Asp Asn Ile 1250 1255 1260 Pro GluLeu Ser Glu Val Val Ile Val Thr Leu Thr Arg Ile Thr 1265 1270 1275 ThrGlu Gly Val Glu Asp Ser Tyr Lys Gly Ala Thr Ile Asp Gln 1280 1285 1290Asp Arg Ser Lys Ser Val Ile Thr Thr Leu Pro Asn Asp Ser Pro 1295 13001305 Phe Gly Leu Val Gly Trp Arg Ala Ala Ser Val Phe Ile Arg Val 13101315 1320 Ala Glu Pro Lys Glu Asn Thr Thr Thr Leu Gln Leu Gln Ile Ala1325 1330 1335 Arg Asp Lys Gly Leu Leu Gly Asp Ile Ala Ile His Leu ArgAla 1340 1345 1350 Gln Pro Asn Phe Leu Leu His Val Asp Asn Gln Ala ThrGlu Asn 1355 1360 1365 Glu Asp Tyr Val Leu Gln Glu Thr Ile Ile Ile MetLys Glu Asn 1370 1375 1380 Ile Lys Glu Ala His Ala Glu Val Ser Ile LeuPro Asp Asp Leu 1385 1390 1395 Pro Glu Leu Glu Glu Gly Phe Ile Val ThrIle Thr Glu Val Asn 1400 1405 1410 Leu Val Asn Ser Asp Phe Ser Thr GlyGln Pro Ser Val Arg Arg 1415 1420 1425 Pro Gly Met Glu Ile Ala Glu IleMet Ile Glu Glu Asn Asp Asp 1430 1435 1440 Pro Arg Gly Ile Phe Met PheHis Val Thr Arg Gly Ala Gly Glu 1445 1450 1455 Val Ile Thr Ala Tyr GluVal Pro Pro Pro Leu Asn Val Leu Gln 1460 1465 1470 Val Pro Val Val ArgLeu Ala Gly Ser Phe Gly Ala Val Asn Val 1475 1480 1485 Tyr Trp Lys AlaSer Pro Asp Ser Ala Gly Leu Glu Asp Phe Lys 1490 1495 1500 Pro Ser HisGly Ile Leu Glu Phe Ala Asp Lys Gln Val Thr Ala 1505 1510 1515 Met IleGlu Ile Thr Ile Ile Asp Asp Ala Glu Phe Glu Leu Thr 1520 1525 1530 GluThr Phe Asn Ile Ser Leu Ile Ser Val Ala Gly Gly Gly Arg 1535 1540 1545Leu Gly Asp Asp Val Val Val Thr Val Val Ile Pro Gln Asn Asp 1550 15551560 Ser Pro Phe Gly Val Phe Gly Phe Glu Glu Lys Thr Val Ser 1565 15701575 151 20 DNA Homo sapiens 151 gtgacaattg cagcctctga 20 152 20 DNAHomo sapiens 152 agtgatattg gcgctcgtct 20 153 20 DNA Homo sapiens 153cttcacctct ggcaacatca 20 154 20 DNA Homo sapiens 154 acttttcccatgaggccttt 20 155 80 DNA Homo sapiens 155 tcatggaact ctgtctccagtgactttgca ttggaacata gactctgatc ctgatggtga 60 tctcgccttc acctctggca 80156 80 DNA Homo sapiens 156 ttgggcagac gagcgccaat atcactgtgg agatattgcctgacgaagac ccagaactgg 60 ataaggcatt ctctgtgtca 80 157 18 DNABacteriophage SP6 157 atttaggtga cactatag 18 158 20 DNA Bacteriophage T7158 taatacgact cactataggg 20 159 33 DNA Homo sapiens 159 cccaagcttatgcaggcgct taacattacc ccg 33 160 32 DNA Homo sapiens 160 cgggatccttaatgccactg tctaaaggaa ga 32 161 68 DNA Homo sapiens 161 cgggatccttacttgtcgtc gtcgtccttg tagtccatat gcccactgtc taaaggagaa 60 ttctcaac 68162 39 DNA Homo sapiens 162 gcagcagcgg ccgcctcttt tcaaaaagtt gttccttgg39 163 37 DNA Homo sapiens 163 gcagcagtcg acataataat aacacactaa gggatac37 164 37 DNA Homo sapiens 164 gcagcagcgg ccgcatggaa ggactctttt caaaaag37 165 36 DNA Homo sapiens 165 gcagcagtcg acgggatact tacttgaacg actctg36 166 19 DNA Homo sapiens 166 gcagacgagc gccaatatc 19 167 25 DNA Homosapiens 167 gacacagaga atgccttatc cagtt 25 168 28 DNA Homo sapiens 168tgggtcttcg tcaggcaata tctccaca 28 169 17 PRT Homo sapiens 169 Val SerThr Glu Arg Cys Leu Ser Val Leu Trp Pro Ile Trp Tyr Arg 1 5 10 15 Cys170 16 PRT Homo sapiens 170 His Leu Ser Ala Val Val Cys Val Leu Leu TrpAla Leu Ser Leu Leu 1 5 10 15 171 103 PRT Homo sapiens 171 Ala Asp PheLeu Phe Leu Cys Phe Gln Ile Ile Asn Cys Leu Val Tyr 1 5 10 15 Leu SerAsn Phe Phe Cys Ser Ile Ser Ile Asn Phe Pro Ser Phe Phe 20 25 30 Thr ThrVal Met Thr Cys Ala Tyr Leu Ala Gly Leu Ser Met Leu Ser 35 40 45 Thr ValSer Thr Glu Arg Cys Leu Ser Val Leu Trp Pro Ile Trp Tyr 50 55 60 Arg CysArg Arg Pro Arg His Leu Ser Ala Val Val Cys Val Leu Leu 65 70 75 80 TrpAla Leu Ser Leu Leu Leu Ser Ile Leu Glu Gly Lys Phe Cys Gly 85 90 95 PheLeu Phe Ser Asp Gly Asp 100 172 19 PRT Homo sapiens 172 Thr Ile Leu LeuThr Val Leu Val Phe Leu Leu Cys Gly Leu Pro Phe 1 5 10 15 Gly Ile Gln173 17 PRT Homo sapiens 173 Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr PhePhe Val Gly Ser Phe 1 5 10 15 Arg 174 76 PRT Homo sapiens 174 Met AspSer Thr Ile Pro Val Leu Gly Thr Glu Leu Thr Pro Ile Asn 1 5 10 15 GlyArg Glu Glu Thr Pro Cys Tyr Lys Gln Thr Leu Ser Phe Thr Gly 20 25 30 LeuThr Cys Ile Val Ser Leu Val Ala Leu Thr Gly Asn Ala Val Val 35 40 45 LeuTrp Leu Leu Gly Cys Arg Met Arg Arg Asn Ala Val Ser Ile Tyr 50 55 60 IleLeu Asn Leu Val Ala Ala Asp Phe Leu Phe Leu 65 70 75 175 49 PRT Homosapiens 175 Phe Asp Phe Ile Thr Ala Ala Trp Leu Ile Phe Leu Phe Met ValLeu 1 5 10 15 Cys Gly Ser Ser Leu Ala Leu Leu Val Arg Ile Leu Cys GlySer Arg 20 25 30 Gly Leu Pro Leu Thr Arg Leu Tyr Leu Thr Ile Leu Leu ThrVal Leu 35 40 45 Val 176 58 PRT Homo sapiens 176 Leu Leu Cys Gly Leu ProPhe Gly Ile Gln Trp Phe Leu Ile Leu Trp 1 5 10 15 Ile Trp Lys Asp SerAsp Val Leu Phe Cys His Ile His Pro Val Ser 20 25 30 Val Val Leu Ser SerLeu Asn Ser Ser Ala Asn Pro Ile Ile Tyr Phe 35 40 45 Phe Val Gly Ser PheArg Lys Gln Trp Arg 50 55 177 18 PRT Homo sapiens 177 Pro Ile Leu LysLeu Ala Leu Gln Arg Ala Leu Gln Asp Ile Ala Glu 1 5 10 15 Val Asp 178 20PRT Homo sapiens 178 Leu Thr Pro Ile Asn Gly Arg Glu Glu Thr Pro Cys TyrLys Gln Thr 1 5 10 15 Leu Ser Phe Thr 20 179 27 PRT Homo sapiens 179 LeuThr Gly Asn Ala Val Val Leu Trp Leu Leu Gly Cys Arg Met Arg 1 5 10 15Arg Asn Ala Val Ser Ile Tyr Ile Leu Asn Leu 20 25 180 12 PRT Musmusculus 180 Ile Asn His Tyr Phe Cys Asp Leu Leu Pro Leu Leu 1 5 10 18110 PRT Mus musculus 181 Ser Lys Ala Phe Ser Thr Cys Ser Ser His 1 5 10182 12 PRT Mus musculus 182 Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys AspVal 1 5 10 183 15 PRT Mus musculus 183 Ser Thr Glu Gly Arg Ser Lys AlaPhe Ser Thr Cys Ser Ser His 1 5 10 15 184 14 PRT Mus musculus 184 PhePhe Gly Ser Ala Ala Phe Met Tyr Leu Gln Pro Ser Ser 1 5 10 185 17 PRTHomo sapiens 185 Ile Val Pro Met Leu Asn Pro Leu Ile Tyr Ser Leu Arg AsnLys Asp 1 5 10 15 Val 186 13 PRT Rattus norvegicus 186 Ser Ser Met AspGln Gly Lys Val Ser Ser Val Phe Tyr 1 5 10 187 16 PRT Rattus norvegicus187 Val Pro Met Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Asp Val 1 510 15 188 20 PRT Homo sapiens 188 Ile Thr Val Glu Ile Leu Pro Asp GluAsp Pro Glu Leu Asp Lys Ala 1 5 10 15 Phe Ser Val Ser 20 189 18 PRT Homosapiens 189 Leu Ser Val Ser Ser Gly Ser Leu Gly Ala His Ile Asn Ala ThrLeu 1 5 10 15 Thr Val 190 77 PRT Homo sapiens 190 Ser Asp Asp Pro TyrGly Ile Phe Ile Phe Ser Glu Lys Asn Arg Pro 1 5 10 15 Val Lys Val GluGlu Ala Thr Gln Asn Ile Thr Leu Ser Ile Ile Arg 20 25 30 Leu Lys Gly LeuMet Gly Lys Val Leu Val Ser Tyr Ala Thr Leu Asp 35 40 45 Asp Met Glu LysPro Pro Tyr Phe Pro Pro Asn Leu Ala Arg Ala Thr 50 55 60 Gln Gly Arg AspTyr Ile Pro Ala Ser Gly Phe Ala Leu 65 70 75 191 13 PRT Homo sapiens 191Gly Ala Asn Gln Ser Glu Ala Thr Ile Ala Ile Ser Ile 1 5 10 192 87 PRTHomo sapiens 192 Lys Gly Leu Met Gly Lys Val Leu Val Ser Tyr Ala Thr LeuAsp Asp 1 5 10 15 Met Glu Lys Pro Pro Tyr Phe Pro Pro Asn Leu Ala ArgAla Thr Gln 20 25 30 Gly Arg Asp Tyr Ile Pro Ala Ser Gly Phe Ala Leu PheGly Ala Asn 35 40 45 Gln Ser Glu Ala Thr Ile Ala Ile Ser Ile Leu Asp AspAsp Glu Pro 50 55 60 Glu Arg Ser Glu Ser Val Phe Ile Glu Leu Leu Asn SerThr Leu Val 65 70 75 80 Ala Lys Val Gln Ser Arg Ser 85

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence selected from the groupconsisting of: (a) a polynucleotide fragment of SEQ ID NO:7 or apolynucleotide fragment of the cDNA sequence included in ATCC DepositNo: ______, which is hybridizable to SEQ ID NO:7; (b) a polynucleotideencoding a polypeptide fragment of SEQ ID NO:20 or a polypeptidefragment encoded by the cDNA sequence included in ATCC Deposit No:______, which is hybridizable to SEQ ID NO:7; (c) a polynucleotideencoding a polypeptide domain of SEQ ID NO:20 or a polypeptide domainencoded by the cDNA sequence included in ATCC Deposit No: ______, whichis hybridizable to SEQ ID NO:7; (d) a polynucleotide encoding apolypeptide epitope of SEQ ID NO:20 or a polypeptide epitope encoded bythe cDNA sequence included in ATCC Deposit No: ______, which ishybridizable to SEQ ID NO:7; (e) a polynucleotide encoding a polypeptideof SEQ ID NO:20 or the cDNA sequence included in ATCC Deposit No:______, which is hybridizable to SEQ ID NO:7, having GPCR activity; (f)an isolated polynucleotide comprising nucleotides 172 to 1152 of SEQ IDNO:7, wherein said nucleotides encode a polypeptide corresponding toamino acids 2 to 328 of SEQ ID NO:20 minus the start codon; (g) anisolated polynucleotide comprising nucleotides 1 to 1152 of SEQ ID NO:7,wherein said nucleotides encode a polypeptide corresponding to aminoacids 1 to 328 of SEQ ID NO:20 including the start codon; (h) apolynucleotide which represents the complimentary sequence (antisense)of SEQ ID NO:7; (i) (a) a polynucleotide fragment of SEQ ID NO:10 or apolynucleotide fragment of the cDNA sequence included in ATCC DepositNo: ______, which is hybridizable to SEQ ID NO:10; (j) a polynucleotideencoding a polypeptide fragment of SEQ ID NO:23 or a polypeptidefragment encoded by the cDNA sequence included in ATCC Deposit No:______, which is hybridizable to SEQ ID NO:10; (k) a polynucleotideencoding a polypeptide domain of SEQ ID NO:23 or a polypeptide domainencoded by the cDNA sequence included in ATCC Deposit No: ______, whichis hybridizable to SEQ ID NO:10; (l) a polynucleotide encoding apolypeptide epitope of SEQ ID NO:23 or a polypeptide epitope encoded bythe cDNA sequence included in ATCC Deposit No: ______, which ishybridizable to SEQ ID NO:10; (m)a polynucleotide encoding a polypeptideof SEQ ID NO:23 or the cDNA sequence included in ATCC Deposit No:______, which is hybridizable to SEQ ID NO:10, having GPCR activity; (n)an isolated polynucleotide comprising nucleotides 21 to 950 of SEQ IDNO:10, wherein said nucleotides encode a polypeptide corresponding toamino acids 2 to 311 of SEQ ID NO:23 minus the start codon; (o) anisolated polynucleotide comprising nucleotides 18 to 950 of SEQ IDNO:10, wherein said nucleotides encode a polypeptide corresponding toamino acids 1 to 311 of SEQ ID NO:23 including the start codon; (p) apolynucleotide which represents the complimentary sequence (antisense)of SEQ ID NO:10; (q) (a) a polynucleotide fragment of SEQ ID NO:13 or apolynucleotide fragment of the cDNA sequence included in ATCC DepositNo: PTA-3949, which is hybridizable to SEQ ID NO:13; (r) apolynucleotide encoding a polypeptide fragment of SEQ ID NO:26 or apolypeptide fragment encoded by the cDNA sequence included in ATCCDeposit No: PTA-3949, which is hybridizable to SEQ ID NO:13; (s) apolynucleotide encoding a polypeptide domain of SEQ ID NO:26 or apolypeptide domain encoded by the cDNA sequence included in ATCC DepositNo: PTA-3949, which is hybridizable to SEQ ID NO:13; (t) apolynucleotide encoding a polypeptide epitope of SEQ ID NO:26 or apolypeptide epitope encoded by the cDNA sequence included in ATCCDeposit No: PTA-3949, which is hybridizable to SEQ ID NO:13; (u) apolynucleotide encoding a polypeptide of SEQ ID NO:26 or the cDNAsequence included in ATCC Deposit No: PTA-3949, which is hybridizable toSEQ ID NO:13, having GPCR activity; (v) an isolated polynucleotidecomprising nucleotides 26 to 906 of SEQ ID NO:13, wherein saidnucleotides encode a polypeptide corresponding to amino acids 2 to 295of SEQ ID NO:26 minus the start codon; (w) an isolated polynucleotidecomprising nucleotides 23 to 950 of SEQ ID NO:13, wherein saidnucleotides encode a polypeptide corresponding to amino acids 1 to 295of SEQ ID NO:26 including the start codon; (x) a polynucleotide whichrepresents the complimentary sequence (antisense) of SEQ ID NO:13; and(y) a polynucleotide capable of hybridizing under stringent conditionsto any one of the polynucleotides specified in (a)-(x), wherein saidpolynucleotide does not hybridize under stringent conditions to anucleic acid molecule having a nucleotide sequence of only A residues orof only T residues.
 2. The isolated nucleic acid molecule of claim 1,wherein the polynucleotide fragment consists of a nucleotide sequenceencoding a human G-protein coupled receptor.
 3. A recombinant vectorcomprising the isolated nucleic acid molecule of claim
 1. 4. Arecombinant host cell comprising the vector sequences of claim
 3. 5. Anisolated polypeptide comprising an amino acid sequence selected from thegroup consisting of: (a) a polypeptide fragment of SEQ ID NO:20 or theencoded sequence included in ATCC Deposit No: ______; (b) a polypeptidefragment of SEQ ID NO:20 or the encoded sequence included in ATCCDeposit No: ______, having GPCR activity; (c) a polypeptide domain ofSEQ ID NO:20 or the encoded sequence included in ATCC Deposit No:______; (d) a polypeptide epitope of SEQ ID NO:20 or the encodedsequence included in ATCC Deposit No: ______; (e) a full length proteinof SEQ ID NO:20 or the encoded sequence included in ATCC Deposit No:______; (f) a polypeptide comprising amino acids 2 to 328 of SEQ IDNO:20, wherein said amino acids 2 to 328 comprising a polypeptide of SEQID NO:20 minus the start methionine; (g) a polypeptide comprising aminoacids 1 to 328 of SEQ ID NO:20; (h) a polypeptide fragment of SEQ IDNO:23 or the encoded sequence included in ATCC Deposit No: ______; (i) apolypeptide fragment of SEQ ID NO:23 or the encoded sequence included inATCC Deposit No: ______, having GPCR activity; (j) a polypeptide domainof SEQ ID NO:23 or the encoded sequence included in ATCC Deposit No:______; (k) a polypeptide epitope of SEQ ID NO:23 or the encodedsequence included in ATCC Deposit No: ______; (l) a full length proteinof SEQ ID NO:23 or the encoded sequence included in ATCC Deposit No:______; (m) a polypeptide comprising amino acids 2 to 311 of SEQ IDNO:23, wherein said amino acids 2 to 311 comprising a polypeptide of SEQID NO:23 minus the start methionine; (n) a polypeptide comprising aminoacids 1 to 311 of SEQ ID NO:23; (o) a polypeptide fragment of SEQ IDNO:26 or the encoded sequence included in ATCC Deposit No: PTA-3949; (p)a polypeptide fragment of SEQ ID NO:26 or the encoded sequence includedin ATCC Deposit No: PTA-3949, having GPCR activity; (q) a polypeptidedomain of SEQ ID NO:26 or the encoded sequence included in ATCC DepositNo: PTA-3949; (r) a polypeptide epitope of SEQ ID NO:26 or the encodedsequence included in ATCC Deposit No: PTA-3949; (s) a full lengthprotein of SEQ ID NO:26 or the encoded sequence included in ATCC DepositNo: PTA-3949; (t) a polypeptide comprising amino acids 2 to 295 of SEQID NO:26, wherein said amino acids 2 to 295 comprising a polypeptide ofSEQ ID NO:26 minus the start methionine; and (u) a polypeptidecomprising amino acids 1 to 295 of SEQ ID NO:26;
 6. The isolatedpolypeptide of claim 5, wherein the full length protein comprisessequential amino acid deletions from either the C-terminus or theN-terminus.
 7. An isolated antibody that binds specifically to theisolated polypeptide of claim
 5. 8. A recombinant host cell thatexpresses the isolated polypeptide of claim
 5. 9. A method of making anisolated polypeptide comprising: (a) culturing the recombinant host cellof claim 8 under conditions such that said polypeptide is expressed; and(b) recovering said polypeptide.
 10. The polypeptide produced by claim9.
 11. A method for preventing, treating, or ameliorating a medicalcondition, comprising the step of administering to a mammalian subject atherapeutically effective amount of the polypeptide of claim 5, or amodulator thereof.
 12. A method of diagnosing a pathological conditionor a susceptibility to a pathological condition in a subject comprising:(a) determining the presence or absence of a mutation in thepolynucleotide of claim 1; and (b) diagnosing a pathological conditionor a susceptibility to a pathological condition based on the presence orabsence of said mutation.
 13. A method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectcomprising: (a) determining the presence or amount of expression of thepolypeptide of claim 5 in a biological sample; and (b) diagnosing apathological condition or a susceptibility to a pathological conditionbased on the presence or amount of expression of the polypeptide.
 14. Anisolated nucleic acid molecule consisting of a polynucleotide having anucleotide sequence selected from the group consisting of: (a) apolynucleotide encoding a polypeptide of SEQ ID NO:20; (b) an isolatedpolynucleotide consisting of nucleotides 172 to 1152 of SEQ ID NO:7,wherein said nucleotides encode a polypeptide corresponding to aminoacids 2 to 328 of SEQ ID NO:20 minus the start codon; (c) an isolatedpolynucleotide consisting of nucleotides 169 to 1152 of SEQ ID NO:7,wherein said nucleotides encode a polypeptide corresponding to aminoacids 1 to 328 of SEQ ID NO:20 including the start codon; (d) apolynucleotide encoding the Gene 7 polypeptide encoded by the cDNA clonecontained in ATCC Deposit No. ______; (e) a polynucleotide whichrepresents the complimentary sequence (antisense) of SEQ ID NO:7; (f) apolynucleotide encoding a polypeptide of SEQ ID NO:23; (g) an isolatedpolynucleotide consisting of nucleotides 21 to 950 of SEQ ID NO: 10,wherein said nucleotides encode a polypeptide corresponding to aminoacids 2 to 311 of SEQ ID NO:23 minus the start codon; (h) an isolatedpolynucleotide consisting of nucleotides 18 to 950 of SEQ ID NO: 10,wherein said nucleotides encode a polypeptide corresponding to aminoacids 1 to 311 of SEQ ID NO:23 including the start codon; (i) apolynucleotide encoding the Gene 10 polypeptide encoded by the cDNAclone contained in ATCC Deposit No. ______; (j) a polynucleotide whichrepresents the complimentary sequence (antisense) of SEQ ID NO:10; (k) apolynucleotide encoding a polypeptide of SEQ ID NO:26; (l) an isolatedpolynucleotide consisting of nucleotides 26 to 906 of SEQ ID NO: 13,wherein said nucleotides encode a polypeptide corresponding to aminoacids 2 to 295 of SEQ ID NO:26 minus the start codon; (m) an isolatedpolynucleotide consisting of nucleotides 23 to 906 of SEQ ID NO: 13,wherein said nucleotides encode a polypeptide corresponding to aminoacids 1 to 295 of SEQ ID NO:26 including the start codon; (n) apolynucleotide encoding the Gene 13 polypeptide encoded by the cDNAclone contained in ATCC Deposit No. PTA-3949; (o) a polynucleotide whichrepresents the complimentary sequence (antisense) of SEQ ID NO:13; 15.The isolated nucleic acid molecule of claim 14, wherein thepolynucleotide comprises a nucleotide sequence encoding a humanG-protein coupled receptor.
 16. A recombinant vector comprising theisolated nucleic acid molecule of claim
 15. 17. A recombinant host cellcomprising the recombinant vector of claim
 16. 18. An isolatedpolypeptide consisting of an amino acid sequence selected from the groupconsisting of: (a) a polypeptide fragment of SEQ ID NO:20 having GPCRactivity; (b) a polypeptide domain of SEQ ID NO:20 having GPCR activity;(c) a full length protein of SEQ ID NO:20; (d) a polypeptidecorresponding to amino acids 2 to 328 of SEQ ID NO:20, wherein saidamino acids 2 to 328 consisting of a polypeptide of SEQ ID NO:20 minusthe start methionine; (e) a polypeptide corresponding to amino acids 1to 328 of SEQ ID NO:20; (f) a polypeptide encoded by the cDNA containedin ATCC Deposit No. ______; (g) a polypeptide fragment of SEQ ID NO:23having GPCR activity; (h) a polypeptide domain of SEQ ID NO:23 havingGPCR activity; (i) a full length protein of SEQ ID NO:23; (j) apolypeptide corresponding to amino acids 2 to 311 of SEQ ID NO:23,wherein said amino acids 2 to 311 consisting of a polypeptide of SEQ IDNO:23 minus the start methionine; (k) a polypeptide corresponding toamino acids 1 to 311 of SEQ ID NO:23; (l) a polypeptide encoded by thecDNA contained in ATCC Deposit No. ______; (m)a polypeptide fragment ofSEQ ID NO:26 having GPCR activity; (n) a polypeptide domain of SEQ IDNO:26 having GPCR activity; (o) a full length protein of SEQ ID NO:26;(p) a polypeptide corresponding to amino acids 2 to 295 of SEQ ID NO:26,wherein said amino acids 2 to 295 consisting of a polypeptide of SEQ IDNO:26 minus the start methionine; (q) a polypeptide corresponding toamino acids 1 to 295 of SEQ ID NO:26; (r) a polypeptide encoded by thecDNA contained in ATCC Deposit No. PTA-3949;
 19. The method forpreventing, treating, or ameliorating a medical condition of claim 11,wherein the medical condition is selected from the group consisting of aneural disorder; an endocrine disorder; a sleep disorder; disorders thataffect the nucleus accumbens, disorders that affect the brains ‘rewardcenter’ function, neurotransmitter release disorders, disordersaffecting the release of dopamine, disorders affecting the release ofopioid peptides, disorders affecting the release of serotonin, disordersaffecting the release of GABA, pineal gland disorders, disordersaffecting the establishment of circadian rhythms, disorders affectingthe maintenance of circadian rhythms, disorders affecting the control ofthe sleep/wake cycle; melatonin secretion disorders, pituitary hormonesecretion disorders, oxytocin secretion disorders, disorders affectingneuroendocrine response to stressful stimuli, disorders affectingoxytocin secretion during neuroendocrine response to stressful stimuli,disorders affecting nocturnal patterns of hormone secretion, disordersaffecting the nocturnal hormone secretion of prolactin, disordersaffecting the nocturnal hormone secretion of cortisol, and/or disordersaffecting the nocturnal hormone secretion of growth hormone;neuro-pathologies, including responses to stress, and propensity todevelop addictive behaviors, as well as a vast number of neuroendocrineabnormalities including sleep disorders; and brain tumors.